Advanced Dairy Chemistry

5 Higher Order Structures of the Caseins: A Paradox? 183 Biopolymers: Molecules, Clusters, Networks, and Ingber, D.E. (1998). The architecture of life. Scientific Interactions, M.L. Fishman, P.X. Qi and L. Wicker, American. 278(1), 48–57. eds., American Chemical Society, Washington, DC. pp. 1–18. Kumosinski, T.F. and Farrell, H.M., Jr. (1991). Calcium- Farrell, H.M., Jr., Qi, P.X. and Uversky V.N. (2006c). New induced associations of the caseins: thermodynamic views of protein structure: applications to the caseins: linkage of calcium binding to colloidal stability of protein structure and functionality, in, Advances in casein micelles. J. Protein Chem. 10, 3–16. Biopolymers: Molecules, Clusters, Networks, and Interactions, M.L. Fishman, P.X. Qi and L. Wicker, Kumosinski, T.F. and Farrell, H.M., Jr. (1994). Solubility eds., American Chemical Society, Washington, DC. of proteins: protein-salt-water interactions, in, Protein pp 52–70. Functionality in Food Systems, N.S. Hettiarachchy Farrell, H.M., Jr., Malin, E.L., Brown, E.M. and Mora- and G.R. Ziegler, eds., Marcel Dekker, Inc., New York, Gutierrez A. (2009). Review of the chemistry of aS2- pp. 39–77. casein and the generation of a homologous molecular model to explain its properties. J. Dairy Sci. 92, Kumosinski, T.F., Pessen, H., Farrell, H.M., Jr. and 1338–1353. Brumberger, H. (1988). Determination of the quater- Garnier, J., Osguthorpe, D.J. and Robson, B. (1978). nary structural states of bovine casein by small-angle Analysis of the accuracy and implications of simple X-ray scattering: submicellar and micellar forms. methods for predicting the secondary structure of Arch. Biochem. Biophys. 266, 548–561. globular proteins. J. Mol. Biol. 120, 97–120. Graham, E.R.B., Malcolm, G.M. and McKenzie, H.A. Kumosinski, T.F., Brown, E.M. and Farrell, H.M., Jr. (1984). On the isolation and conformation of bovine (1993a) Three dimensional molecular modeling of b-casein A1. Int. J. Biol. Macromol. 6, 155–161. bovine caseins: an energy-minimized b-casein struc- Groves, M.L., Dower, H.J. and Farrell, H.M., Jr., (1992). ture. J. Dairy Sci., 76, 931–945. Reexamination of the polymeric distributions of k-casein isolated from bovine milk. J. Protein Chem. Kumosinski, T.F., Brown, E.M. and Farrell, H.M., Jr. 11, 21–28. (1993b). Three dimensional molecular modeling of Groves, M.L., Wickham, E.D. and Farrell, H.M., Jr., bovine caseins: an energy minimized k-casein struc- (1998). Environmental effects on disulfide bonding ture. J. Dairy Sci. 76, 2507–2520. patterns of bovine k-casein. J. Protein Chem. 17, 73–84. Kumosinski, T.F., Brown, E.M. and Farrell, H.M., Jr. Halfmann, R. and Lindquist, S. (2010). Epigenetics in the (1994a). Predicted energy minimized aS1-casein extreme: prions and the inheritance of environmen- working model, in, Molecular Modeling from tally acquired traits. Science 330, 629–632. Virtual Tools to Real Problems, T.F. Kumosinski Haque, Z., Kristjansson, M. and Kinsella, J.E. (1987). and M.N. Liebman, eds., ACS Symposium Series Interaction between k-casein and b-lactoglobulin: 576. American Chemical Society, Washington, DC. possible mechanism. J. Agric. Food Chem. 35, pp. 368–390. 644–649. Hoagland, P.D., Unruh, J.J., Wickham, E.D. and Farrell, Kumosinski, T.F., King, G. and Farrell, H.M., Jr. (1994b). H.M., Jr. (2001). Secondary structure of bovine aS2- An energy minimized casein submicelle working casein: theoretical and experimental approaches. model. J. Protein Chem. 13, 681–700. J. Dairy Sci. 84, 1944–1949. Holt, C. and Sawyer, L. (1993). Caseins as rheomorphic Kumosinski, T.F., King, G. and Farrell, H.M., Jr. (1994c). proteins: interpretation of primary and secondary Comparison of the three dimensional molecular mod- structures of the alpha-s1-caseins, beta-caseins and els of bovine submicellar caseins with small-angle kappa-caseins. J. Chem. Soc. Faraday Trans. 89, X-ray scattering. Influence of protein hydration. 2683–2692. J. Protein Chem. 13, 701–714. Horne, D.S. (1998). Casein interactions: casting light on the black boxes, the structure in dairy products. Int. Kumosinski, T.F., Uknalis, J.J., Cooke, P.H. and Farrell, Dairy J. 8, 171–177. H.M., Jr. (1996). Correlation of refined models for Horne, D.S. (2006). Casein micelle structure: models and casein submicelles with electron microscopic studies muddles. Curr. Opi. Coll. & Inter. Sci. 11, 146–153. of casein. Lebens. Wiss. Technol. 29, 326–333. Hummer, G., Garde, S., Garcia, A.E., Paulaitis, M.E. and Pratt, L.R. (1998). The pressure dependence of hydro- Le Parc, A., Leonil, J. and Chanat, E. (2010). aS1-Casein, phobic interactions is consistent with the observed which is necessary for efficient ER-Golgi casein trans- pressure denaturation of proteins. Proc. Nat. Acad. port, is also present in a tightly membrane associated Sci. USA. 95, 1552–1555. form. BMC Cell Biology 11, 65. Huq, N.L., Cross, K.J. and Reynolds, E.C. (1995). A 1H NMR study of the casein phosphopeptide aS1-casein Malin, E.L., Brown, E.M., Wickham, E.D. and Farrell, (59–79). Biochim. Biophys. Acta. 1247, 201–208. H.M., Jr. (2005). Contributions of terminal peptides to the associative behavior of aS1-casein. J. Dairy Sci. 88, 2318–2328. Mora-Gutierrez, A., Kumosinski, T.F. and Farrell, H.M., Jr. (1997). 17O NMR studies of bovine and caprine casein hydration and activity in deuterated sugar solu- tions. J. Agr. Food Chem. 45, 4545–4553. Niewold, T.A., Murphy, C.L., Hulskamp-Koch, C.A., Tooten, P.C. and Gruys, E. (1999). Casein related amyloid, characterization of a new and unique amy- loid protein isolated from bovine corpora amylacea. Amyloid: Int. J. Exp. Clin. Invest. 6, 244–249.

184 H.M. Farrell Jr et al. Onuchic, J.N., Nymeyer, A.E., Garcia, A.E., Chahine, J. Swaisgood, H.E. (1982) Chemistry of milk proteins, in, and Socci, N.D. (2000). The energy landscape theory Developments in Dairy Chemistry, Vol. 1, P.F. Fox, of protein folding: insights into folding mechanisms ed., Applied Science Publishers, London, pp. 1–60. and scenarios. Adv. Protein Chem. 53, 87–152. Syme, C.D., Blanch, E.W., Holt, C., Jakes, R., Goedert, Palmer, D.S., Christensen, A.U., Sorensen, J., Celik, L., M., Hecht, L. and Barron, L.D. (2002). A Raman opti- Qvist, K.B. and Schiott, B. (2010) Bovine chymosin: a cal activity study of rheomorphism in caseins, synu- computational study of recognition and binding of cleins and tau. New insights into the structure and bovine k-casein. Biochemistry. 49, 2563–2573. behavior of natively unfolded proteins. Eur. J. Biochem. 269, 148–156. Paulsson, M. and Dejmek, P. (1990). Thermal denatur- ation of whey proteins in mixtures with caseins stud- Thorn, D.C., Meehan, S., Sunde, M., Rekas, A., Gras, ied by differential scanning calorimetry. J. Dairy Sci. S.L., MacPhee, C.E., Dobson, C.M., Wilson, M.K. 73, 590–600. and Carver, J.A. (2005). Amyloid fibril formation by bovine milk k-casein and its inhibition by the molecu- Pepper, L. (1972). Casein interactions as studied by gel lar chaperones aS- and b-casein. Biochemistry 44, chromatography and ultracentrifugation. Biochim. 17027–17036. Biophys. Acta. 278, 147–154. Thorn, D.C., Ecroyd, H., Sunde, M., Poon, S. and Carver, Pepper, L. and Farrell, H.M., Jr. (1982). Interactions lead- J.A. (2008). Amyloid fibril formation by bovine milk ing to the formation of casein submicelles. J. Dairy aS2-casein occurs under physiological conditions yet Sci. 65, 2259–2266. is prevented by its natural counterpart aS1-casein. Biochemistry 47, 3926–3936. Qi, P.X., Wickham, E.D., Piotrowski, E.G., Faegerquist, C.K., and Farrell, H.M., Jr. (2005). Implication of Thurn, A., Buchard, W. and Niki, R. (1987). Structure of C-terminal deletion on the structure and stability of casein micelles II. aS1-casein. Coll. Polym. Sci. 265, bovine b-casein. Protein J. 24, 431–444. 897–902. Schmidt, D.G. (1982). Association of caseins and casein Tompa, P. (2002). Intrinsically unstructured proteins. micelle structure, in, Developments in Dairy Chemistry, Trends Biochem. Sci. 27, 527–533. P.F. Fox, ed., Applied Science Publishers Ltd., London, pp. 61–86. Tompa, P. and Kalmar L. (2010). Power law distribution defines structural disorder as a structural element directly Schmidt, D.G. and Payens, T.A.J. (1976). Micellar aspects linked with function. J. Mol. Biol. 403, 346–350. of casein, in, Surface and Colloid Science, Vol. 9, E. Matijevic, ed., John Wiley and Sons, New York, pp. Tunick, M.H., Cooke, P.H., Malin E.L., Smith, P.W. and 165–229. Holsinger, V.H. (1997). Reorganization of casein sub- micelles in Mozzarella cheese during storage. Int. Slattery, C.W. and Evard, R. (1973). A model for the for- Dairy J. 7, 149–155. mation and structure of casein micelles from subunits of variable composition. Biochim. Biophys. Acta. 317, Uversky, V.N. (2002). Natively unfolded proteins: a point 529–538. where biology waits for physics. Protein Science 11, 739–756. Snoeren, T., van Markwijk, H.M.B. and van Montfort R. (1980). Some physical chemical properties of bovine Waugh, D.F. (1970). Formation and structure of casein aS2-casein. Biochim. Biophys. Acta. 622, 268–276. micelles, in, Milk Proteins Chemistry and Molecular Biology Vol. 2, H.A. McKenzie, ed., Academic Press, Sood, S.M., Lekic, T., Jhawar, H., Farrell, H.M., Jr. and New York, London, pp. 3–85. Slattery, C.W. (2006). Reconstituted micelle forma- tion using reduced, carboxymethylated bovine k-casein Xie, D., Bhakuni, V. and Freire, E. (1991). Calorimetric and human b-casein. Protein J 25, 352–360. determination of the energetics of the molten globule intermediate in protein folding: apo-a-lactalbumin. Steinbacher, S., Seckler, R., Miller, S., Steipe, B., Huber, Biochemistry. 30, 10673-10678. R. and Reinemer, P. (1994). Crystal structure of P22 tailspike protein: interdigitated subunits in a thermo- stable trimer. Science 265, 383–386.

Casein Micelle Structure, Functions, 6 and Interactions D.J. McMahon and B.S. Oommen 6.1 Introduction Brown 1984; Rollema 1992; Holt 1992; Holt and Horne 1996; Walstra 1999; de Kruif and Holt Casein micelles are particles of colloidal size that 2003). Recent insightful evaluations of the vari- can be described as supramolecules or a system ous models of casein micelle structure and the consisting of multiple molecular entities held process by which this supramolecule is assem- together and organized by means of non-covalent bled can be found in Farrell et al. (2006a), Horne intermolecular binding interactions. The exis- (2006) and Dalgleish (2011). In this chapter, we tence of such a colloidal particle consisting of a aim to discuss the supramolecular structure of mixture of calcium phosphate stabilized by cal- casein micelles on the basis of investigations cium-insoluble proteins has long been recognized using electron microscopy (McMahon and (Linderstrøm Lang 1929). These supramolecules McManus 1998; Oommen 2004; McMahon and serve as the prime nutritional source of calcium, Oommen 2008), interpreted in terms of their phosphate, and amino acids to meet the growth known physical and chemical attributes. The term and energy requirements of mammalian neonates “casein micelle” has been used in a generic sense and have the biological function of transporting for the calcium-phosphate-protein colloidal par- calcium phosphate without calcification through ticles in milk for many years, but it is now very the mammary milk system (Horne 2002a, b; de apparent that the supramolecular structure of Kruif and Holt 2003). Numerous models have these colloidal particles varies depending on pH, been proposed to explain the supramolecular cooling, heating, and addition of other ingredi- structure of casein micelles and these have been ents to milk. We have reserved use of casein reviewed repeatedly (e.g., Bloomfield and Morr micelle as a descriptor for these supramolecules 1973; Farrell 1973; Garnier 1973; Swaisgood and as they are synthesized and secreted from the Brunner 1973; Thompson and Farrell 1973; mammary gland, i.e., native casein micelles in Slattery 1976; Schmidt 1980; McMahon and non-cooled raw milk. The model structure described in McMahon and Oommen (2008) was D.J. McMahon (*) based on observations of casein micelles from Western Dairy Center, Utah State University, bovine milk although it appears that casein Logan, UT, USA micelles from milk of other species are similar. e-mail: [email protected] In bovine milk, the caseins consist of four B.S. Oommen major proteins, as1-casein, as2-casein, b-casein, Glanbia Nutritionals Research, and k-casein that are secreted in their numerous Twin Falls, ID, USA genetic and posttranslational variations (Chaps. 4 and 15). The calcium-sensitive caseins (as1-, as2-, P.L.H. McSweeney and P.F. Fox (eds.), Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects, 185 4th Edition, DOI 10.1007/978-1-4614-4714-6_6, © Springer Science+Business Media New York 2013

186 D.J. McMahon and B.S. Oommen and b-caseins) are members of a single-gene biologically important in binding to surfaces and family as seen from the homologous gene the formation of macroscopic networks. sequences of different species while the k-casein Maintaining such flexibility helps explain (de gene is homologous to g-fibrinogen (Swaisgood Kruif and Holt 2003) the slightly nutritionally 1992). Caseins undergo posttranslational phos- suboptimal amino acid composition of the phorylation to varying degrees at seryl residues Ca-sensitive caseins since their evolutionary (see Chap. 4). imperative is preventing pathological precipita- tion of calcium phosphate in the mammary gland. The secondary structure of the caseins has This structural flexibility of the casein molecules often been referred to as random coil, although is then inherently passed on to the supramolecule this is misleading and caseins as a group of pro- level and can be observed in the response of teins can be expected to be as highly adapted to casein micelles to different environments. their biological function as any other structural Whether considered as being rheomorphic or protein (de Kruif and Holt 2003). A better having a molten globule structure, conforma- description is to consider the caseins as being tional flexibility of the caseins seems critical for intrinsically unstructured proteins (Farrell et al., the casein supramolecule to maintain its struc- 2006b; Chap. 5) similar to other secretory ture. Various structures for the casein micelle are Ca-binding proteins (Smith et al., 2004). Their possible based on how they are synthesized and physiological function in the mammary gland chemical treatments to which they are subjected. results from their different partially folded con- Structural information presented in this chapter formations and from structural transitions will primarily be focused on the supramolecular between them. Other terms used to describe the organization of native casein micelles. considerable conformational flexibility of the caseins include molten globule structure (Malin 6.2 Modeling the Casein Micelle et al., 2005) and rheomorphic structure (Holt and Sawyer 1993). It is interesting to look at the evolution of models of casein micelles as our understanding and abil- Farrell et al. (2006b) predicted that as1- and ity to analyze casein systems has increased. The as2-caseins are natively unfolded proteins with core-coat model proposed by Waugh and Noble extended coil-like (or pre-molten globule-like) (1965) consisted of spherical particles of as1- and conformations, whereas b- and k-caseins would b-caseins with k-casein as the coat. The model possess molten globule-like properties. Proteins put forward by Payens (1966) showed a consider- in a molten globule state have a compact struc- able portion of k-casein located on the periphery ture with a high degree of hydration and side of the casein micelle and compactly folded as- chain flexibility; they possess native secondary caseins attached to loose b-caseins as the core, structures with little tertiary folds. Because of with calcium ions interacting with phosphate or their lack of a fixed three-dimensional tertiary carboxylic acid groups of the proteins. Bloomfield conformation, caseins can react very rapidly to and Morr (1973) postulated the existence of a environmental changes. This ability to exist in size-determining supramolecule framework pre- various conformations was described by Holt and dominantly made of as1-casein with b- and Sawyer (1993) as the caseins being rheomorphic. k-caseins attached to the framework and filling its In the mammary cells, caseins function by seques- interstices through Ca bridges. This was based on tering small clusters of calcium phosphate, thus partial calcium depletion in which b- and k-ca- preventing precipitation and calcification of the seins dissociate leaving a framework having the mammary milk synthesis and transport system same frictional resistance as the original supra- (Horne 2002a; de Kruif and Holt 2003). This molecule. Rose (1969) proposed a model in which conformational flexibility further allows them to end-to-end b-casein association initiated supra- interact with multiple target molecules, and in molecule formation to which as- and k-casein that sense, caseins can be considered as part of the scavenger class of unfolded proteins (Smith et al., 2004). Such conformational flexibility is

6 Casein Micelle Structure, Functions, and Interactions 187 molecules were in turn bound to form a protein Fig. 6.1 Transmission electron micrograph of a casein aggregate. In the presence of calcium, these were micelle obtained from bovine milk that was been treated cross-linked by calcium phosphate to form the with glutaraldehyde followed by poly-l-lysine immobili- supramolecule. Garnier and Ribadeau-Dumas zation onto a parlodion-coated copper grid, then stained (1970) proposed a model based on the aggrega- with uranyl oxalate, rapidly frozen and freeze-dried. tion behavior of the different caseins with trimers Reprinted from J. Dairy Sci. 91:1709–1721 with permis- of k-casein acting as the branching nodes and as- sion from American Dairy Science Association and b-caseins as the branches to form a porous supramolecule. subunits. However, none of these models could explain the dissociation behavior of casein supra- At about the same time, and on the basis of molecules on treatment with excess k-casein or sedimentation behavior of urea- and oxalate- urea (Holt 1998). treated casein micelles, Morr (1967) put forth the subunit model in which the subunit core was Support for a supramolecular structure of made of a b-as-casein complex surrounded by an casein micelles consisting of subunits had come as-k-casein complex with the subunits held from chemical and physical measurements of together by calcium and colloidal calcium phos- casein micelles and their dissociation into smaller phate linkages. Schmidt and Payens (1976) particles, by analysis of particles (~20-nm diam- modified this model by proposing subunits with a eter) made from sodium caseinate, and their sub- hydrophobic core surrounded by a hydrophilic sequent growth into larger supramolecules coat of carboxylic and phosphate groups. As pro- (~100- to 20-nm diameter) upon addition of cal- posed by Morr (1967), this model also included cium. Early electron micrographs generally calcium, magnesium, and colloidal calcium phos- showed a corpuscular and globular appearance of phate groups as the linkage between the subunits. casein micelles but more recent work has shown Slattery and Evard (1973) proposed that k-casein that the supramolecular structure of the casein was localized on particular regions of the submi- micelle is quite sensitive to its environment such cellar surface, thus forming two distinct regions that artifacts are easily introduced during sample which are hydrophilic and hydrophobic. preparation (McMahon and McManus 1998). Aggregation of the subunits occurred by hydro- When viewed using a transmission electron phobic bonding until the whole supramolecule microscope, casein micelles that have been surface was covered with k-casein. However, this glutaraldehyde-fixed appear quite electron dense, model did not include any function of colloidal compact, with an internal quaternary structure calcium phosphate in the stability of the casein that could not be distinguished (Fig. 6.1). This is supramolecule. even more problematic when using an electron microscopy method that involves metal coating. Schmidt (1980) and later Walstra (1990) The additive nature of glutaraldehyde fixation has improved on this model by postulating that those the potential to fill interstices between proteins surface regions of the submicelles not covered with polymerized glutaraldehyde. Such material with k-casein consisted of polar moieties of other would appear transparent using transmission caseins, e.g., their phosphoserine residues. The subunits aggregated together via the colloidal calcium phosphate attached to as1-, as2-, and b-caseins. As in the model suggested by Slattery and Evard (1973), casein supramolecular growth terminated when the colloidal particle surface was covered with k-casein. Walstra (1999) modified the submicellar model of casein micelles to include calcium phosphate packages to be placed not only on the surface of submicelles as the bridging between them but also inside the

188 D.J. McMahon and B.S. Oommen electron microscopy (TEM) compared to proteins vesicles as branched and linear chains (Farrell et al., 2006a). This, however, is only slightly (or calcium phosphate) that have been stained larger than the presumed size of monomeric casein (i.e., Stokes radius ~4 nm) and smaller than with heavy metals. In comparison, any metal their polymeric forms (e.g., native k-casein poly- mers with Stokes radius of ~10 nm) (Swaisgood coating used in scanning electron microscopy or 2003). In similar studies of lactating mammary glands, Helminen and Ericsson (1968) observed replica formation would cover both proteins and that branching fibrils of protein (8–9 nm thick) initially formed in the Golgi vacuoles, then accu- cross-linked glutaraldehyde. The ion beam etch- mulated to form, what they termed, the mosaic pattern of the casein micelles, and were excreted ing observations of Hojou et al. (1977) that into the lumen. Such fibril formation provides greater support for a framework rather than sub- strongly supported the submicelle model can then unit model of the casein micelle supramolecule. be explained as the ion bombardment etching While the subunit model was widely accepted in which clustering of casein molecules into small away the accumulated glutaraldehyde and allow- subunits which then further clustered to form the secreted colloidal particle (McMahon and Brown ing a metal coating to be applied directly to pro- 1984; Rollema 1992), such a particulate internal structure of casein micelles has not been observed tein chains. (McMahon and McManus 1998; Holt et al., 2003; Dalgleish et al., 2004). Rather, observations dur- A polyelectrolyte brush model of casein ing dissociation experiments and scattering data of simulated casein micelles provide the most micelles in which the caseins randomly associate support for presence of submicelles. Such experi- ments do not usually include microstructural to form an entangled structure within which nano- observations in addition to measurement of phys- icochemical parameters. From small-angle X-ray clusters of calcium phosphate are held has also scattering, Kumosinski et al. (1988) studied the supramolecular structure of casein micelles syn- been proposed (Holt 1992; Holt and Horne 1996). thesized by adding calcium to sodium caseinate and deduced that the submicelles consisted of a The binding of caseins to pre-crystalline calcium spherical hydrophobic core with a loose hydro- philic shell with the submicelles being held phosphate nanoclusters prevents their nucleation together through calcium phosphate linkages with overlap of hydrophilic regions of the pro- and growth and thus blocks calcification of the teins in adjacent submicelles. mammary tissue. In this model, the binding of McMahon and McManus (1998) argued that previous electron microscopic images of casein caseins to calcium phosphate initiates the forma- micelles do not represent the native casein micelle supramolecule as the sample preparation needed tion of the casein supramolecule (de Kruif and in most electron microscopy introduces artifacts into the imaged structure. A more representative Holt 2003). However, as described by Horne image of casein micelles was that they comprise strands of electron-dense regions, no more than (2006) these two processes of protein polymer- 8–10 nm in length. Variations in scattering inten- sities from X-ray and neutron scattering would ization and calcium binding of casein molecules would occur simultaneously. Farrell et al. (2006a) proposed that the forma- tion of small casein aggregates during casein supramolecule synthesis within Golgi vesicles occurs prior to formation of calcium phosphate nanoclusters. This was based on the sequence of events occurring during bioassembly of casein micelles in the mammary gland. In this scenario, the caseins are synthesized on ribosomes of the rough endoplasmic reticulum and then undergo folding and association (both self and mixed asso- ciation are possible) that helps them escape deg- radation. Transportation via vesicles at the Golgi apparatus then occurs in which Farrell et al. (2006a) propose that the sequence of events is (1) an increase in calcium concentration in the vesi- cles, (2) phosphorylation of the caseins when the calcium concentration is sufficiently high (kinase K ~20 mM), and (3) further secretion of calcium M phosphate into the vesicles. The protein aggre- gates were reported to be spherical complexes of about 10 nm diameter that were present in the

6 Casein Micelle Structure, Functions, and Interactions 189 then not imply an internal repeating substructure Fig. 6.2 Low magnification transmission electron micro- but rather a heterogeneous internal structure. graph of field of view of casein micelles obtained from Pignon et al. (2004) concluded that X-ray scatter- bovine milk by poly-l-lysine immobilization onto a parlo- ing by casein micelles could be attributed to glob- dion-coated copper grid that was then stained with uranyl ular supramolecules (with radius of gyration of oxalate, rapidly frozen, and freeze-dried. Reprinted from J. 102 nm) and the open conformational structure Dairy Sci. 91:1709–1721 with permission from American of the supramolecules’ constituent proteins (with Dairy Science Association radius of gyration of 5–6 nm) rather than from any globular submicelles. Dalgleish (2011) con- number or weight average diameter (Udabage cluded that the various X-ray and small-angle et al., 2003). Using a number distribution yields neutron scattering data could be best interpreted a median diameter of 1.1 × 102 nm while a weight as being a result of calcium phosphate nanoclus- distribution yields a median diameter of ters rather than varying density of protein. 2.2 × 102 nm. The presence of some casein A polycondensation model proposed by Horne micelles being up to 7 × 102 nm in diameter (1998) envisaged cross-linking of individual underlies the broad size distribution (de Kruif caseins through hydrophobic regions of the 1998) typically observed (Fig. 6.2) for casein caseins and bridging involving clusters of cal- micelles. Although when considered on a num- cium phosphate for the assembly of the casein ber basis, the particle-size distribution can be supramolecule. This was later described as a represented by a log-normal distribution dual-binding model (Horne 2002a) in which (Udabage et al., 2003). growth of hydrophobically bonded proteins is inhibited by electrostatic repulsive interactions, Early measurements of casein micelle size whereby total interaction energy can be consid- (Schmidt et al., 1973) had also reported particles ered to be the sum of electrostatic repulsion and less than 20 nm in diameter accounting for 80% hydrophobic interaction. According to this model, of particles by number (but only 3% by volume). k-casein is linked into the casein supramolecule More recent measurements (de Kruif 1998; by hydrophobic bonding of its N-terminal region Udabage et al., 2003) infer that ~50 nm diameter and, therefore, further growth beyond the k-casein is the lower end of particle-size distribution, and is not possible as it does not possess either a large numbers of very small casein micelles have phosphoserine cluster for linkage via colloidal not been observed in electron microscopic images calcium phosphate or another hydrophobic of milk (McMahon et al., 2009). Such polydis- anchor point to extend the chain. persity is common in colloidal systems although biological systems tend to be more homodisperse 6.3 Physical Properties (Bloomfield 1979) and is indicative that the physiological function of casein micelles is inde- Casein micelles are highly hydrated and sponge- pendent of particle size. The number of protein like colloidal particles. Of the approximately 4 g water/g protein contained within the supramole- cule, only about 15% is bound to the protein, the remainder being simply occluded within the particle (de Kruif and Holt 2003; Farrell et al., 2003). Supramolecule size distribution had been reported as extending from 20 to 600 nm diame- ter with a median size between 100 and 200 nm (Schmidt et al., 1974; Bloomfield and Mead 1975; de Kruif 1998), depending on whether the method used to measure particle size generates a

190 D.J. McMahon and B.S. Oommen molecules that constitute the casein supramole- actions with the calcium phosphate nanoclusters. cule is about 104 for a colloidal particle of Destabilization of casein micelles near their iso- ~150 nm diameter (Kirchmeuer 1973). Given electric pH shows that ionic interactions are impor- their range in diameter from 50 to 700 nm, the tant for supramolecule stabilization. As milk is structure of casein micelles is such that it allows acidified there is a decrease in magnitude of net for synthesis in the mammary gland of supramo- negative charge on individual protein molecules lecules containing 103 to 106 casein molecules. and the overall net charge of casein supramole- cules (Heertje et al., 1985). Solubilization of cal- 6.4 Thermodynamic Forces cium phosphate also causes calcium phosphate inside the casein supramolecule to be gradually A variety of forces that derive from the chemistry depleted. Any protein molecules (such as some of the caseins (Chap. 4) come into play in pre- as1-, as2-, and b-caseins) that are bound into the serving the structural integrity of casein supra- supramolecule structure only by the sequestering molecules. These include hydrophobic-related action of their phosphoserine groups toward the interactions, ionic and electrostatic interactions, calcium phosphate can dissociate especially if hydrogen bonds, disulfide bonds, and steric stabi- milk is cold and hydrophobic interactions are lization. At low temperatures, the contribution of minimized. entropy to free-energy change becomes less important, allowing proteins that are primarily For casein micelles, steric stabilization is linked via hydrophobic interactions to migrate provided by having an increased proportion of from the supramolecule. b-Casein and k-casein k-casein on the periphery of the colloidal supra- diffuse out of the micelles at low temperatures molecule. The highly hydrophilic glycomacro- more so than as1- and aS2-caseins, suggesting a peptide portion of k-casein (residues 106–169) greater proportion of the b- and k-casein mole- provides stability against aggregation (McMahon cules are held into the lattice structure of the and Brown 1984). When these peripheral protein casein supramolecule solely by hydrophobic segments on separate particles interpenetrate, or interactions. Similar involvement of hydrophobic compress each other upon close approach, there is regions of the proteins has long been recognized a loss of entropy due to restriction in their in casein polymerization with as1/k-casein copo- configurational freedom and a positive free-energy lymers being formed by hydrophobic rather than change from the increased protein segment con- electrostatic interactions (Dosako et al., 1980). A centration. Such steric repulsion is a short-range consequence of interaction of the highly phos- force and depends on magnitude of solvation phorylated caseins (as1- and as2-caseins, and to forces around the protein, and for native casein some extent b-casein) with calcium phosphate micelles, it is sufficiently strong to overcome the nanoclusters is that it would segregate these dispersion force of attraction (Horne 1986; Walstra caseins from the singly phosphorylated k-casein 1990; Holt 1992; Holt and Horne 1996). during synthesis and dissociation. 6.5 Casein Polymerization Many sites for ionic bonding exist within the different caseins that can play a role in casein The polymerization (aggregation) behavior of supramolecule stabilization. In addition to the caseins is based on the possibility of calcium- phosphoserine side chains of the calcium-sensitive mediated interactions via clusters of phosphoser- caseins, there can also be electrostatic interactions ine groups (Dalgleish and Parker 1979), between carboxylate residues and calcium (and interactions via hydrophobic regions, interactions any other divalent metal ions present in milk). with water via their hydrophilic regions These can help impart structural stability to the (Yoshikawa et al., 1981), as well as hydrogen casein supramolecule by providing calcium cross- bonding and the various electrostatic interactions linking between proteins in addition to their inter- (such as calcium bridging between negatively

6 Casein Micelle Structure, Functions, and Interactions 191 charged sites and ion pairing) that are common to mers consisting of blocks with high levels of all proteins. Because of the varied ways in which hydrophobic or high levels of hydrophilic amino the caseins can interact with themselves (homopo- acid residues (Horne 2002a; Euston and Horne lymers and aggregates), with each other (het- 2005). eropolymers and aggregates), and with minerals (e.g., ionic Ca and calcium phosphate nanoclus- As described by Horne (2002b), b-casein can ters), there is a range of functionalities over which act as a duo-block polymer that can interact with they can interact depending on their surrounding other proteins via calcium bridging because of its environment. b-Casein and k-casein form soap- cluster of phosphoserine residues as well through like micelles with a degree of association of 23 its hydrophobic region. as1-Casein could interact and 30, respectively (Payens and Vreeman 1982); as a tri-block polymer through predominantly in the absence of calcium (Swaisgood 2003), as1- hydrophobic regions at its C- and N-terminals, as casein forms tetramers and subsequent linear well as a hydrophilic-rich region that contains its polymers (Payens 1966), while b-casein can form phosphoserine clusters. as2-Casein can be con- linear polymers of indefinite size (Payens and sidered a hybrid of as1-casein and b-casein Markwijk 1963). Both as1- and b-casein form because, starting from its N-terminal, it can inter- mixed complexes with k-casein (Garnier et al., act through two sets of alternating hydrophobic- 1964; Payens 1968; Garnier 1973), and they can rich and hydrophilic-rich regions and thus has the interact and polymerize to different degrees under possibility of acting as a tetra-block polymer. various conditions of pH, ionic strength, and k-Casein would act primarily as a monoblock temperature. unit because it lacks a cluster of phosphoserine resi- dues and the glycosylation of its hydrophilic In the absence of calcium, Thurn et al. (1987) C-terminal prevents any strong electrostatic interac- reported that at high ionic strength, as1-casein tions with other proteins except via its N-terminal forms polymer-like chains with its hydrophobic hydrophobic region. It is known to be present in regions joined end-to-end. Malin et al. (2005) milk as disulfide-linked oligomers with other found predominately dimers for all three genetic k-casein molecules (Rasmussen et al., 1999) and variants of this protein at 37°C and at physiologi- either alone or as an aggregate it can be consid- cal ionic strength. All of the caseins can form ered as a polymerization terminator. Dalgleish some type of self-association structure when in (2011) described the role of k-casein in casein solution, but their association in a mixed system micelle synthesis as being similar to a surfactant- containing calcium phosphate is more complex, limited polymerization reaction in that since as this increases their functionality (f) or number k-casein is not involved in calcium phosphate of possible simultaneous polymer interactions nanocluster stabilization, it is available to limit that can occur. the overall process and accumulates on the supra- molecule periphery. Dalgleish and Parker (1979) assigned an aver- age f = 2 for calcium-induced aggregation of as1- Colloidal size aggregates similar in size to casein based on four to ten calcium ions being casein micelles can be synthesized using as1-, bound per protein molecule. Horne et al. (1988) as2-, b-, and k-caseins, along with calcium, phos- assigned chain-terminating (f = 1), bifunctional phate, and citrate as components (Schmidt et al., (f = 2), and trifunctional (f = 3) roles to k-, b-, and 1974, 1977; Slattery 1979). Though they may as-caseins, respectively, although it may be more seem to be similar to native casein micelles appropriate to assign as1-casein as f = 2 and as2- (Knoop et al., 1979), their structure may vary casein as f = 3. Within casein micelles there are depending on the degree of casein hydration, pH, opportunities for both specific phosphoserine- salt balance, and the forces contributing to its mediated interactions with calcium phosphate integrity. When a sodium caseinate solution was nanoclusters and hydrophobic and ionic interac- examined according to McMahon and Oommen tions with other proteins. These interactions can (2008), the proteins adsorbed onto the parlodion- also be viewed as the caseins being block copoly- coated grids were observed at low magnification

192 D.J. McMahon and B.S. Oommen Fig. 6.3 Low (top) and high (bottom) magnification of is no phosphate present to form calcium phosphate protein aggregates obtained after hydrating skim milk nanoclusters, the calcium would be expected to powder (left), calcium caseinate powder (middle), and be distributed throughout the casein supramolec- sodium caseinate powder (right) using a shear rate ~735 s−1 ular structure and attached to all of the phospho- for 10 min followed by 60 min at 40°C, then processed as serine side chains of the calcium-sensitive described in Fig. 6.2. Reprinted from Oommen (2004) proteins. These phosphoserine sites of calcium bridging between proteins and other negatively to be present as a gel-like structure with the charged sites on the proteins not involved in proteins forming a mesh across the grid surface bridging would then be readily available for and no evidence of spherical colloidal structure exchange during heavy metal staining. (Fig. 6.3). In the absence of calcium, the proteins remained as strands or small agglomerates of Given the open conformation and structure proteins. Interestingly, these are reminiscent of of the caseins, their flexibility and rheomorphic the protein chains observed in Golgi vesicles by response to their chemical environment, it can Farrell et al. (2006a) and Helminen and Ericsson be expected that the internal structure of casein (1968). This is as expected given the polymeriza- supramolecules is dependent on the sequence tion characteristics of the caseins and the translu- of their synthesis. Both sodium caseinate and cent (non-milky) appearance of sodium casein calcium caseinate have acid-precipitated casein solutions. Farrer and Lips (1999) predicted that as a starting material, either as a wet slurry or a self-assembly of caseins in sodium caseinate dried acid casein powder. As discussed in more solutions would produce linear polymeric rods depth below, acidification of milk solubilizes and weakly branched chains. For the same solids calcium phosphate nanoclusters present in concentration, calcium caseinate has a lower vis- native casein micelles and reduces their cosity than sodium caseinate solutions and z-potential such that there is a release followed appears opaque and white, as does milk. by reaggregation of proteins into the colloidal particle that then undergoes coagulation Calcium caseinate solutions contain both large through hydrophobic interactions to form the colloidal particles (approximately 300 nm diam- acid casein gel (McMahon et al., 2009). The eter) and smaller particles of 10 to 20 nm diam- supramolecular structure of such spherical par- eter as observed using TEM (Fig. 6.3). They have ticles formed during acid coagulation of milk is a spherical appearance similar to native casein not expected to be the same as native casein micelles in milk but when stained with heavy micelles. metals (e.g., OsO4) they appear much more elec- tron dense and blacker. This increased electron In manufacture of calcium and sodium casein- density could result from a combination of higher ate, neutralization of the acid gel restores the net concentration of protein in the particles or a high negative charge on the colloidal particle surface, level of calcium binding to the caseins. As there causing the gel to disintegrate as the colloidal particles repel each other. In the absence of cal- cium, the proteins continue to disassemble until a stable state is reached in which short chains are all that remain (see Fig. 6.3). Calcium caseinate can be formed directly by neutralizing the acid curd with calcium hydroxide or by first neutral- izing the acid curd with ammonia (or sodium hydroxide) and then adding calcium (Bylund et al., 2003). This would be similar to adding cal- cium to a sodium caseinate solution. When cal- cium chloride is added to EDTA-calcium-depleted casein micelles, the calcium binding to phospho- serine in such synthetic casein supramolecules is

6 Casein Micelle Structure, Functions, and Interactions 193 different to that which occurs in native casein In artificial casein micelles, about 50% of cal- micelles (Gebhardt et al., 2011). cium is exchanged within 1 min and an additional 20% in 24 h (Pierre and Brule 1981; Pierre et al., Neutralization with calcium hydroxide occurs 1983). The remainder is difficult to exchange more slowly than with sodium hydroxide (Bylund (Zhang et al., 1996), and the nanoclusters are vir- et al., 2003). Simultaneously to increasing the net tually bound into the casein supramolecule at the negative charge, calcium bridging would occur normal pH of milk (de Kruif and Holt 2003). Holt between individual proteins within the colloidal et al. (1998) proposed a core-shell model of cal- particles, keeping them intact and preventing fur- cium phosphate nanoclusters based on stabiliza- ther disintegration of the supramolecular struc- tion by bovine b-casein (f1–25) peptides. The ture. Simulated casein micelles formed in this nanoclusters comprise a spherical core of radius manner would then be expected to be a mass of ~2.4 nm, consisting of ~355 CaHPO4.2H2O units entangled protein chains (similar to that described and surrounded by ~49 peptide chains forming a by Holt and Horne 1996) that are cross-linked tightly packed shell with an outer radius of through numerous calcium bridges predomi- ~4.0 nm. Given the greater bulk of intact caseins nantly occurring via phosphoserine residues of (~4.5-nm radius) and their multiple phosphate as1-, as2-, and b-caseins. However, if the acid clusters, it could be considered that calcium casein is first neutralized with ammonia or sodium phosphate nanoclusters could be stabilized by hydroxide and then calcium added, it would be binding three to five molecules of as1-, as2-, or the casein polymer chains and small particles that b-casein. Calcium phosphate nanoclusters have become the building blocks for making the simu- thus been considered as having polymerization lated casein micelles. These could then be f > 4 (Holt et al., 1996). expected to have subunit attributes as shown by Pessen et al. (1989). For as1-casein, its two phosphate centers (at amino acids 41–51 and 61–70) could act as poten- 6.6 Interactions with Calcium tial regions of association with different nano- Phosphate clusters (de Kruif and Holt 2003) although, as discussed by Horne (2006), these two phosphate Approximately 32 mM of calcium is present in centers on as1-casein are most likely to attach to milk in different forms that are bound (22 mM) or adjacent facets of the same nanocluster. Rapid not bound (10 mM) to the casein supramolecule binding of caseins to calcium phosphate nano- (Bloomfield and Mead 1975). Of the unbound clusters was proposed by Holt et al. (2003) as calcium, only 3 mM are in free ionic form being the structure-forming points for mammary (Bloomfield and Morr 1973). As discussed above, gland synthesis of the casein micelle. This would calcium contained within casein supramolecules produce a calcium phosphate-protein aggregate may be bound directly to phosphate ester and car- in the size range of 7–13 nm which fits the disin- boxyl groups of caseins or as part of the calcium tegration observations by Hojou et al. (1977) and phosphate nanoclusters that in turn are bound to also particles observed in mammary gland Golgi phosphate esters of casein molecules. Calcium vesicles during casein micelle synthesis. phosphate nanoclusters are considered to be an Although, Farrell et al. (2006a) argued that initial acidic calcium phosphate salt that also contains calcium phosphate concentrations in the Golgi magnesium and citrate (de Kruif and Holt 2003) vesicles are too low to allow the formation of and probably zinc (Meyer and Angino 1977), nanoclusters prior to casein aggregation. with at least three phosphoserine phosphates also participating in the nanocluster structure (Aoki With phosphoserine clusters of attached as1-, et al., 1992; de Kruif and Holt 2003). as2-, or b-caseins oriented toward and participat- ing in the calcium phosphate nanocluster, their There is a slow exchange of ions between the hydrophobic domains would be oriented so they serum and protein-bound calcium and phosphate. can interact with other casein molecules. Lateral hydrophobic binding to other caseins that are

194 D.J. McMahon and B.S. Oommen attached to the same calcium phosphate nano- and Oommen 2008), by cryo-TEM of thin vitrified cluster would be analogous to protein orientation films of casein micelles suspension (Marchin et al., and monolayer in situ polymerization that occurs 2007), and by using cryo-transmission electron when proteins are absorbed onto an oil–water tomography (Trejo et al., 2011). When preparing interface (Dickinson and Matsumura 1991). If casein micelles for examination by electron the hydrophobic regions are oriented in a plane microscopy, it is important to realize that the integ- away from the calcium phosphate nanocluster, rity of these supramolecules depends on a combi- hydrophobic interactions and binding could occur nation of factors including strong electrostatic with other casein entities (including monomeric linkages of caseins to calcium phosphate as well as casein, casein homo/heteropolymers and aggre- protein–protein interactions such as H-bonding, gates, and caseins that are part of other calcium salt bridging (via calcium ions), ion pairing, and phosphate-phosphocasein complexes) such as hydrophobic interactions (McMahon and Brown described by the dual-binding approach to casein 1984). Furthermore, the casein molecule confor- supramolecule synthesis of Horne (2002b). The mational flexibility that facilitates their rapid and interaction of caseins with the calcium phosphate accurate response to environmental change—the nanoclusters immobilizes their flexible domains basis for their functioning so well to sequester and and induces rigidity in the supramolecule transport calcium phosphate in the mammary (Rollema and Brinkhuis 1989). If a calcium phos- gland (Horne 2002a)—can easily lead to structural phate nanocluster is dissolved, the surrounding rearrangements during the chemical treatments protein organization would remain intact unless used during sample preparation for electron the hydrophobic and electrostatic interactions microscopy. between them were also disrupted. Such structural changes are known to occur 6.7 Microstructural Imaging during the fixation, alcohol dehydration, and crit- ical point drying steps of sample preparation for The non-crystallizing nature of the individual scanning electron microscopy. This makes inter- caseins, and their aggregates, limits the use of pretation of extracellular structures of biological techniques such as X-ray crystallography and specimens viewed at very high magnification multidimensional proton NMR to study their rather limited because of uncertainties regarding structure. Electron microscopy has thus been an artifact formation resulting from sample prepara- important tool in deciphering the supramolecular tion (e.g., structural changes), surrounding mate- arrangement of the caseins. The challenge has rials (e.g., coatings, surface tension, ice), or been how to prepare and view casein micelles so image capture (e.g., contrast settings). In that the resultant electron micrographs exhibit McMahon and Oommen (2008), a copper grid minimal variation of the casein micelle supramo- covered in nitrocellulose with a poly-l-lysine lecule from its native form (McMahon and coating was used to attach a monolayer of immo- McManus 1998). bilized casein micelles stained with uranyl oxalate, then immersed in liquid N2-cooled Freon Surface images can be obtained using scanning to freeze the sample without causing ice crystal electron microscopy without metal coating formation, following by sublimation to remove (Dalgleish et al., 2004) and cross sections of the the water molecules, and then viewed by TEM internal structure can be seen using TEM of freeze- using a goniometer stage. fractured cryo-protected casein micelle suspen- sions (Heertje et al., 1985; Karlsson et al., 2007). It is typical when using TEM to observe a Total (surface and internal) images can be obtained three-dimensional spherical object such as a by TEM of freeze-dried surface-immobilized casein micelle that the central region of the image casein micelles without resin embedding and sec- is darker than the periphery (Fig. 6.4). This is not tioning (McMahon and McManus 1998; McMahon indicative of a change in electron density but rep- resents more scattering opportunities being pres- ent when the thickness of the sample traversed by

6 Casein Micelle Structure, Functions, and Interactions 195 Fig. 6.4 Casein micelle from bovine milk imaged as described in Fig. 6.2. Reprinted from J. Dairy Sci. 91:1709–1721 with permission from American Dairy Science Association Fig. 6.5 Stereo image of a casein micelle imaged as described in Fig. 6.2 and photographed 8° apart. Reprinted from J. Dairy Sci. 91:1709–1721 with permission from American Dairy Science Association the electron beam is greater. When viewed using greater confidence that what is being observed in stereo pairs of images (Fig. 6.5), this dimensional the micrograph represents a real electron-dense compression artifact can be eliminated and the entity and not an artifact related to imaging at a supramolecular structure is observed to be uni- very high magnification. With recent advances in form throughout the casein micelle. Another instrumentation and software, it is now possible advantage of using stereo pairs is that because to obtain internal structural images of casein multiple images of the same casein micelle are supramolecules at various angles using a 140° obtained at different angles, then only objects stage rotation and then to reconstruct a three- recorded at both angles will converge giving dimensional image (Trejo et al., 2011).

196 D.J. McMahon and B.S. Oommen It must still be recognized that there will be that have a very low electron density and cannot parts of the supramolecule that are more electron be distinguished from the background. dense than others, such as the calcium phosphate nanoclusters, and where there is binding of heavy 6.8 Interlocking Lattice metal ions used to increase contrast. On the pro- Supramolecule teins, this occurs where they carry negative charges (carboxylate and serine phosphate sites) Taking into account a slight underestimation of rather than in hydrophobic regions and where it the volume being occupied by the proteins, a can exchange for calcium in the nanoclusters. model structure of the casein supramolecule was The contrast settings used during image capture developed. When viewing an entire casein micelle and during image manipulation will also influence (see Figs. 6.4 or 6.5), the large number of indi- differentiation of image pixels relating to elec- vidual components being visualized (103 to 106 tron-dense regions and the background (McMahon depending on casein micelle size) makes it and McManus 1998). It must therefore be remem- difficult to view the central region of the supra- bered that only a selective portion of the casein molecule. There are too many overlapping planes molecules are imaged whether this is achieved by of electron-dense locations visually to isolate subjective visual adjustment or by the use of individual planes. Even so, it was evident that computer software. At low contrast (such as in there were no major differences among structural Fig. 6.4), the extension of protein strands outward arrangements between the central portions of on the casein micelle periphery is retained. When casein micelles and their peripheral regions. In viewed using stereo pairs (Fig. 6.5), the predomi- McMahon and Oommen (2008) a region on the nant appearance of casein micelles was that of supramolecule periphery (Fig. 6.6) was selected electron-dense locations present as interlocked for close examination of electron-dense entities chains. Open spaces between the electron-dense and a digitally magnified image of this region locations would represent regions devoid of mat- was then visually examined stereoscopically. ter (i.e., occupied by aqueous serum in the native casein micelle) as well as portions of the proteins Fig. 6.6 A peripheral section of a casein micelle (left) remainder of the electron-dense spots were considered to imaged as described in Fig. 6.2 digitally magnified as a be casein molecules and represented as light grey circles stereo pair and a single plane of electron-dense spots visu- of 8-nm diameter (right). Reprinted from J. Dairy Sci. ally isolated (middle) with intersecting locations desig- 91:1709–1721 with permission from American Dairy nated as calcium phosphate of 4.8 nm diameter, while the Science Association

6 Casein Micelle Structure, Functions, and Interactions 197 When observing a single plane of electron- Fig. 6.7 Schematic cross-sectional diagram of the dense locations, they consisted of short linear and interlocking lattice model of the casein micelle with branched polymer chains that were interlocked casein-calcium phosphate aggregates throughout the together on a regular basis (Fig. 6.6). At these entire supramolecule, branched and linear chains of pro- interlocking sites there was a grouping of tein extending between them, and numerous serum pock- electron-dense locations with many near neigh- ets and channels. Calcium phosphate nanoclusters are bors and the polymer chains appeared to radiate shown with a diameter of 4.8 nm and about 18 nm apart, outward until they encountered another interlock- and caseins shown with hydrodynamic diameter of 8 nm. ing site. Once the skeletal structure of the supra- Reprinted from J. Dairy Sci. 91:1709–1721 with permis- molecule had been determined it was then sion from American Dairy Science Association necessary to make assignments to various loca- tions based on known size and functionalities of ing, and the various electrostatic interactions components that make up the casein micelle. The (such as calcium bridging between negatively most likely candidate for the entities forming the charged sites and ion pairing) that are common to interlocking sites was calcium phosphate nano- all proteins. The observed redundancy in func- clusters because of their ability to bind multiple tionality of caseins in bovine milk suggests that phosphoproteins (i.e., as1-, as2-, and b-casein) and the range of functionalities is more important than these were designated as spheres of 4.8-nm diam- which casein actually performs a particular func- eter as proposed by Holt and Sawyer (1993). Since tion. Also, a molecule such as as2-casein with its it is not possible to differentiate between proteins four potential interaction regions may not always solely on electron microscopic images, the remain- associate with other components to its maximum der of the electron-dense locations were simply functionality because of steric hindrance between assigned as being protein with an average diame- binding partners. Rapid binding of many caseins ter of 8 nm. This gave the appearance of the elec- to calcium phosphate nanoclusters would then act tron-dense material forming into a lattice-type as structure-forming points during casein micelle structure consisting of interlocked orbs with synthesis as proposed by Holt et al. (2003). chains of material that encompassed areas devoid of any electron-dense material (Fig. 6.7). The Further casein molecules can bind to struc- presence of such channels and pools of serum ture-forming aggregates either as monomers, oligo- throughout the interior of casein micelles has been mers, or even as aggregates bound to a different shown recently by Trejo et al. (2011). calcium phosphate nanocluster. Interactions can occur via hydrophobic regions or calcium bridg- With this lattice supramolecule structure ing through their carboxylate or phosphoserine (Fig. 6.7), it is very apparent that casein micelles side chains that are not part of the phosphoserine have a very open, porous structure expected for a colloidal particle with a high voluminosity in which proteins account for only 10–20% of the supramolecule volume. It is also apparent that the amount of water occluded by casein micelles is very dependent on where the surface of the col- loidal supramolecule is placed and the extent of draining that occurs in these peripheral regions. As described above, the polymerization behavior of the various caseins depends on the possibility of calcium-mediated interactions via clusters of phosphoserine groups, the gain in entropy obtained by grouping of hydrophobic regions so as to remove them from the aqueous environment, interactions of hydrophilic regions with water, hydrogen bond-

198 D.J. McMahon and B.S. Oommen clusters bound to the calcium phosphate nano- ters. The distance between interlocking sites clusters (Swaisgood 2003). In the volume appeared similar to the 18 nm interval predicted assigned to each protein molecule, multiple elec- by de Kruif and Holt (2003) for calcium phos- tron-dense locations were often observed, so this phate nanoclusters. Some were further apart assignment is based on what we considered a while others were closer together such that representative arrangement. It should also be there could be from two to six protein mole- realized that the conformational shape of the pro- cules between the interlocking sites. According teins is not spherical and would to some extent to Smith et al. (2004), calcium phosphate depend upon interactions with their neighbors accounts for about 7% of dry mass of casein and can be in the form of compact clusters. micelles, and casein micelles of 200 nm diam- Various structural arrangements, such as long lin- eter and 109 Da contain approximately 800 cal- ear chains, double-stranded chains and short cium phosphate nanoclusters. This equates to a branches on chains, were observed throughout ratio of about 60 protein molecules per calcium casein micelles. This was expected because phosphate nanocluster, which is more than what k-casein, which acts as a chain terminator, is was observed in our electron micrographs if it present throughout the entire casein micelle and is assumed that each interlocking point in the not just on its surface. lattice structure is a calcium phosphate nano- cluster. Some of the interlocking points may The casein micelle supramolecule can thus be also result from branches in the protein chain, considered as an interlocked lattice (Fig. 6.7) in possibly by as2-casein. which the casein molecules both surround the calcium phosphate nanoclusters and extend as 6.9 Modifying the Supramolecule short chains between the interlocking points and outward at the particle periphery. The supramo- Modification of the native physicochemical envi- lecular structure is irregular and allows for a large ronment of milk, such as acidification, heating, diversity of linkages between the proteins includ- blending with other fluid foods, and hydrolysis, ing chain extenders (b-casein or as1-casein), is an integral part of manufacture of various dairy chain branch points (as1-casein or as2-casein), products. Understanding their influence on the chain terminators (k-casein), and interlocking supramolecular structure of casein micelles is points (calcium phosphate nanoclusters). On the vital in tailoring the characteristics and quality of periphery of the casein micelle, there can be some dairy products. chains of proteins extending outward, placing k-casein (either individually or as disulfide-linked 6.9.1 Cooling of Milk polymers) well out from the bulk of the casein micelle. The number of these protuberances is In TEM examination of thin sections of agar- less than postulated in early depictions of the embedded milk at different temperatures casein micelle surface as a hairy layer (or poly- (McMahon et al., 2009), there appeared to be electrolyte brush) on a hard sphere (Holt and very few casein supramolecules at 40°C that were Horne 1996) but extend further into the surround- <80 nm diameter (Fig. 6.8). At lower tempera- ing serum. There is no distinct hairy layer as later tures (20 and 30°C) there appeared to be more recognized (de Kruif and Holt 2003) and some electron-dense material that was only loosely protuberances were observed (McMahon and attached to the supramolecules although no Oommen 2008) to be up to about 30 nm in length, extensive dissociation of the supramolecules which is similar in length to that observed by was observed. At 30°C, the supramolecules were Dalgleish et al. (2004). spherical in shape but many of them had surface protuberances, and in some cases the surface Overall, this supramolecular structure would produce a very stable colloidal particle com- prising many thousands of protein molecules and hundreds of calcium phosphate nanoclus-

6 Casein Micelle Structure, Functions, and Interactions 199 Fig. 6.8 Transmission electron micrographs of thin sec- from J. Dairy Sci. 92:5854–5867 with permission from tions of skim milk that had been glutaraldehyde-fixed and American Dairy Science Association agar-solidified at 40°C (left) and 10°C (right). Reprinted material seemed only loosely attached to the rest Fig. 6.9 Serum proteins obtained from the supernatant of the colloidal particle. At 20°C, the supramol- after centrifuging cold raw milk at 27,000 × g for 2 h and ecule surface had a more tendrillar appearance imaged as described in Fig. 6.2. Reprinted from Oommen with electron-dense areas protruding from the (2004) particle surface. chains and clusters, thus forming a gel. The phys- At 10°C, the supramolecules had a ragged ical properties of such a gel are influenced by appearance, and there was a greater proportion of temperature, and when cold (~4°C), the milk can smaller particles, including some that were no be acidified without gelation occurring. Calcium longer spherical (Fig. 6.8). Also, there were some removal from native casein supramolecule ini- particles that had an absence of material in their tially dissociates weakly bound b- and k-caseins core. Compared to casein micelles at 40°C, the without any apparent change in supramolecule supramolecules at 10°C were less electron dense size (Bloomfield and Morr 1973; Lin et al., 1972). (i.e., there was less heavy metal staining), their Acidification releases as-, b-, and k-caseins from peripheral edges were less distinct with a rela- the supramolecule in varying proportions depend- tively open structure, and there was more protein material dispersed as loose aggregates among the colloidal supramolecules. When milk was cooled to 5°C and centrifuged (27,500 × g for 2 h), the supernatant predominantly contained small pro- tein aggregates about 10–20 nm in size and numerous linear and branched chains of proteins (Fig. 6.9). 6.9.2 Acidification of Milk Upon acidification of milk, it is known that casein supramolecules undergo changes based on charge neutralization (Davies et al., 1977; Kalab et al., 1976) and other factors (Heertje et al., 1985; Holt and Horne 1996; Lucey 2002) that ultimately result in their aggregating into a network of

200 D.J. McMahon and B.S. Oommen Fig. 6.10 Transmission electron micrographs of thin sec- fixed and agar-solidified. Reprinted from J. Dairy Sci. tions of skim milk acidified to pH 5.2 at 40°C (left) and 92:5854–5867 with permission from American Dairy 10°C (right) using glucono-d-lactone then glutaraldehyde- Science Association ing on the experiment being conducted. b-Casein sediment smaller casein supramolecules and has been reported to be preferentially solubilized large aggregates. McMahon et al. (2009) pro- (Snøeren et al., 1984; van Hooydonk et al., 1986) posed that the first phase of acid gelation of milk while others (Roefs et al., 1985; Dalgleish and involves a temperature-dependent dissociation of Law 1988; Singh et al., 1996a) reported equal proteins from the casein supramolecules with dissociation of b- and k-caseins and a lower per- less protein being released when the milk is warm centage of as-caseins. The proteins with higher (Fig. 6.10). At cold temperatures (e.g., 10°C) dis- levels of phosphorylation dissociate less readily sociated proteins were also present as loosely from the casein supramolecule when calcium entangled aggregates. At 40°C, there appears to phosphate is solubilized (Aoki et al., 1988). be sufficient hydrophobic interactions to main- tain the proteins as (small) spherical colloidal Dalgleish and Law (1988) observed that at particles (many with diameters <50 nm). The for- 30°C, dissociation of casein upon acidification of mation of such small spherical particles would milk to pH 5.5 had a constant proportion of as1-, require rearrangement and consolidation of the b-, and k-casein, suggesting that these proteins supramolecule interior structure with a predomi- may have dissociated as an intact complex. The nance of k-casein expected to remain on the smallness of these particles also agrees with them periphery. As calcium phosphate nanoclusters being enriched in k-casein, and having less as1- that interlock the protein strands within the casein casein than the original casein supramolecules. supramolecule are solubilized, the structural This would be expected as k-casein is needed to integrity of the casein supramolecule is weak- stabilize the particles because neither b-casein ened, promoting rearrangements inside the supra- nor as-caseins can exist as monomers under these molecule as well as dissociation. It can thus be conditions (van Hooydonk et al., 1986). expected that the casein supramolecules would undergo a transition to compensate for the loss of At low temperature there is more dissociation interactions with calcium phosphate nanoclus- of proteins from the casein supramolecules dur- ters. New calcium bridging and other electrostat- ing acidification than at higher temperatures. ics protein-protein interactions would occur and Dalgleish and Law (1988) reported 30% and 55% hydrophobic interactions at higher temperatures. being dissociated at 20°C and 4°C, respectively. Singh et al. (1996b) reported only 7% and 22% When milk was acidified at 40°C and casein dissociation at 22°C and 5°C, respectively, but micelles captured using the method of McMahon they used higher centrifugal force that would

6 Casein Micelle Structure, Functions, and Interactions 201 Fig. 6.11 Casein supramolecules from pasteurized skim prepared and imaged as described in Fig. 6.2. Reprinted milk (pH 6.7) and from the same milk acidified with glu- from Oommen (2004) cono-d-lactone and sampled at pH 5.9, 5.4, and 5.1 and and Oommen (2008), increased amounts of elec- stant during the initial acidification of milk, but tron-dense particles were observed around their McMahon et al. (2009) observed an apparent periphery; there was a loss of supramolecule decrease in size of the casein supramolecules integrity and shape, and large tendril appendages with many more small casein supramolecules originated from the supramolecule and extended being present at pH 5.7. This corresponds to the into the surrounding area (Fig. 6.11). By pH 5.9 pH at which Dalgleish and Law (1988) observed the number of surface tendrils increased suggest- maximum dissociation of casein from the supra- ing a progressive breakup of the supramolecule’s molecules at 30°C. However, the measurement of lattice structure from its periphery into the center. dissociated casein is actually a measure of There were also more smaller electron-dense nonsedimentable protein and this is dependent on nonspherical objects (ranging in size from 10 to the centrifugal force applied and whether the pro- 100 nm) in the background that correspond to the teins that are released from the casein micelle loosely entangled protein aggregates observed by remain in monomeric form or polymerize into McMahon et al. (2009) that had adsorbed to the aggregates. When large aggregates, such as the grid in addition to the casein supramolecules. loosely entangled proteins observed by McMahon et al. (2009) are formed, they could be sedimented As the pH was lowered, some loosely entan- and incorrectly presumed to still be part of the gled nonspherical aggregates were observed casein supramolecules. (McMahon et al., 2009) to be as large as the native casein supramolecules. Casein micelle particle McMahon et al. (2009) observed considerable size has been reported (Singh et al., 1996a; loosely entangled protein aggregates present in Dalgleish et al., 2004) to remain relatively con- milk at 30°C that was acidified to pH 5.7 as well

202 D.J. McMahon and B.S. Oommen as many supramolecules in the 50 to 100 nm pasteurized skim milk acidified at 35–50°C starts range. At 40°C and the same pH, the loosely at pH 5.1–5.2 (Kim and Kinsella 1989). When entangled protein aggregates were not evident milk was sampled at pH 5.1, clusters of casein but there were many small supramolecules in the supramolecules were observed (Fig. 6.11) even 30 to 50 nm range. Oommen (2004) also observed though the milk was still fluid. This was because no dissociated material observed around the casein these clusters can adsorb onto the grid using the supramolecules at pH 5.4 (Fig. 6.11). At low method of McMahon and McManus (1998) rather magnification, the supramolecules were well dis- than being restricted to a single plane observed persed and in various sizes while at high using freeze etching or thin sectioning. In such magnification, small clumps of electron-dense cases, chains of particles or gel networks are material (10 to 20 nm diameter) were observed observed only if they reside within the plane of throughout their interior. Between pH 6.7 and pH the section (or the fracture surface). Chains of 5.4, the dominant influences of milk acidification particles that cross the imaged plane may be on protein dissociation and colloidal particle for- observed as a single supramolecule or as a short mation are a balance of temperature-dependent chain depending on the angle at which they cross hydrophobic effects, calcium phosphate solubili- the plane and the thickness of the section (Kalab zation (which is both pH and temperature depen- et al., 1976). The casein supramolecules at pH dent), and charge neutralization. At higher 5.1 (Fig. 6.11) were compact and sufficiently temperature, the hydrophobic effect is sufficient electron dense that internal structure could not be to favor polymerization into spherical colloidal determined, suggesting they are more like the particles rather than remaining as loosely entan- supramolecules in calcium caseinate and have a gled aggregates. different structure to casein micelles as they exist in milk at pH 6.7 and 40°C. Around pH 5.4, soluble b-casein, along with other dissociated proteins, may precipitate at The compact nature of the particles observed their combined isoelectric point (Heertje et al., at pH 5.1 suggests that the re-association of 1985) resulting in the formation of the compact caseins with the residual calcium-depleted casein aggregates of proteins observed within the casein results in a colloidal particle that not only has a supramolecules. As pH drops further, b-casein z-potential approaching zero but whose surface (theoretical pI of 5.26 at 20°C) present in such lacks tendrils of protein that extend into the sol- clusters would become positively charged and vent water. Such supramolecules would lack the provide an attraction with other casein molecules steric repulsion of native casein supramolecules in the supramolecules that are still net negatively (Tuiner and de Kruif 2002) generated by such charged. This would alter peripheral regions of peripheral tendrils. This combined with posi- the supramolecules, and as pH is lowered further tively charged proteins on parts of the supramol- all the protein polymers and aggregates would re- ecule periphery would allow van der Waal’s associate into the compact particles observed at attraction to overwhelm repulsive forces during pH 5.1 (Fig. 6.11). It may be construed that the colloidal particle collisions leading to aggrega- structure of casein supramolecules at the gelation tion and subsequent gelation. pH results from the combined influence of elec- trostatic interactions, hydrophobic interactions, 6.9.3 Calcium Sequestration and calcium bridging among the various caseins. This differs from native casein micelles in which Sequestering of calcium and dissolution of the the interactions between the caseins and calcium calcium phosphate nanoclusters by agents such as phosphate nanoclusters are a predominant con- EDTA can result in disintegration of the native tributor to its internal structure. casein supramolecules (Lin et al., 1972; Aoki et al., 1986; Holt et al., 1986). If calcium is At 40°C, the start of milk gelation was depleted by dialyzing milk against simulated milk observed at pH 5.05 (Oommen 2004) which was similar to previous reports that coagulation of

6 Casein Micelle Structure, Functions, and Interactions 203 Fig. 6.12 Calcium-depleted casein supramolecules in Fig. 6.2. Reprinted from Oommen (2004) and from J. obtained after adding 43 m mol/L EDTA to bovine milk at Dairy Sci. 91:1709–1721 with permission from American 40°C (left) and 4°C (middle and right) imaged as described Dairy Science Association ultrafiltrate containing EDTA, the hydrodynamic ionic calcium <2 m mol/L, protein dissociation radius of those particles remains constant up to a occurred while the calcium phosphate nanoclus- critical level of ionic calcium reduction (Bloomfield ters remained intact within the casein micelle, and Morr 1973). Direct addition of EDTA into and Holt et al. (1986) concluded that the milk causes some casein micelles to dissociate retention of proteins within the calcium-reduced completely while the rest of them still remain casein supramolecule was related to their phos- intact apparently depending on the extent of cal- phoserine content, i.e., as2- > as1- > b- > k-casein. cium dissociation. When calcium in milk was che- lated using EDTA, the casein micelles were Adding ionic calcium via dialysis causes observed to dissociate partially and clusters of transfer of soluble casein into the supramolecule thin tentacles and strands of proteins were formed without any apparent change in size of the casein (Fig. 6.12). At 40°C there were many small micelles (Bloomfield and Morr 1973). Thus, the filamentous aggregates that appeared to contain supramolecule structure appears sufficiently three to six filigreed rings of protein. In cold milk dynamic that it retains its overall structure even (5°C) there were clusters of particles (15–50 nm when some proportion of proteins are added or in size) and linear strands of protein that were removed. When casein micelles are dissociated approximately 1–2 nm in width and up to 30 nm by calcium removal, the resultant particles more long. These chains appeared to contain branching closely resembled sodium caseinate than small points or overlapped other chains. The remaining synthetic casein micelles (Rollema and Brinkhuis casein micelles lacked regularity in shape or size 1989). It appears that this is the common struc- and were of low electron density. Disintegration ture of non-colloidal caseins, such as those of the casein supramolecule into smaller particles obtained upon cooling of milk (Fig. 6.9), in the was considered as strong supporting evidence for absence of calcium and manufacture of sodium the submicelle model. However, caseins form a caseinate (Fig. 6.3) or after chelation of calcium dynamic system and, therefore, can rearrange by EDTA (Fig. 6.12). themselves after disintegration. They do not exist as monomers under physiological conditions 6.9.4 Heating of Milk (Swaisgood 1992) so it would be expected they would remain as small aggregates. Relatively severe milk temperature changes are involved in the manufacture of many dairy prod- Dialysis of casein micelles against a phos- ucts. Heat treatment of milk above 70°C results in phate-free buffer also causes supramolecule dis- denaturation of b-lactoglobulin and its subse- sociation and releasing predominantly b- and quent interaction with other denatured whey pro- k-caseins (Holt et al., 1986) with less as1- and teins and with caseins via disulfide, hydrophobic, as2-caseins. When the dialysis buffer was satu- and ionic interactions. Factors such as pH, salt rated in colloidal calcium phosphate but with

204 D.J. McMahon and B.S. Oommen Fig. 6.13 Casein supramolecules and other proteins in milk adjusted to pH 6.4 (left), pH 6.7 (middle), and pH 7.0 (right) then heated to 90°C for 30 min and imaged as in Fig. 6.2. Reprinted from Oommen (2004) system, and ionic strength and the presence of gesting that some dissociation had occurred and solvents and other solutes can affect such interac- there were compact and electron-dense regions as tions with the casein supramolecules during heat- well as numerous tendrils of attached protein ing. Understanding the mechanism and the influence chains and clusters of lower electron density. of these treatments on structural changes of casein is vital in tailoring the characteristics and quality When pH was increased to 7.0 and the milk of dairy products. Concomitantly with heat-in- then heated to 90°C for 30 min, there were large duced protein interactions, the pH of milk (100 to 500 nm diameter) spherical electron- decreases as calcium and phosphate becomes less dense particles (casein supramolecules) with soluble and further associate with the casein large nonspherical aggregates of lower electron supramolecules releasing H+. density either attached to the casein supramole- cules or occupying the surrounding areas. The Associations of b-lactoglobulin with k-casein distinct internal filigreed ring-like lattice struc- through disulfide linkages is also pH dependent ture of the casein supramolecule observed in (Heertje et al., 1985; Corredig and Dalgleish native casein micelles was still apparent. 1996; Anema and Klostermeyer 1997). Anema and Li (2003) reported an increase in casein 6.9.5 Addition of Ethanol supramolecule size by 25–30 nm at pH 6.5 when heated at 90°C for 30 min compared to an increase In the presence of ethanol, the milk protein sys- of 5–10 nm at pH 6.7. Electron microscopic tem undergoes destabilization (Horne 1992). This investigation of this heat-induced complex forma- had been considered a function of alcohol reduc- tion by Heertje et al. (1985) showed that at higher ing the dielectric constant causing a collapse of pH (pH ³ 7.0), large aggregates formed that were the brush-like C-terminal region of k-casein on not attached to the casein supramolecules, while the casein supramolecule periphery. With the at lower pH (pH £ 6.7) the aggregates were open interlocked lattice structure proposed by attached around their periphery. At pH 6.7 after McMahon and Oommen (2008), this change in heat treatment to 90°C, Oommen (2004) observed dielectric constant would be experienced by all casein supramolecules as dark electron-dense proteins in the supramolecule. This would initiate particles with numerous appendages around their a global contraction and rearrangement causing a periphery (Fig. 6.13). These appendages were of collapse and partial dissociation of the casein various sizes and shapes and had the appearance supramolecules (Fig. 6.14). Horne and Davidson of large, but less electron-dense, protein aggre- (1987) reported that the dissociated particles in a gates attached to proteins on the casein supramo- 1:1 trifluoroethanol/milk mixture were approxi- lecules similar to Heertje et al. (1985). Heating mately the same hydrodynamic size as in native milk that had been acidified to pH 6.4 produced milk, but were dissimilar in molecular weight clumps of casein supramolecules attached to a and sedimentation properties. Contrary to this virtual web of aggregated material. Their electron explanation, O’Connell et al. (2001a), using confo- density was less than that observed at pH 6.7 sug-

6 Casein Micelle Structure, Functions, and Interactions 205 Fig. 6.14 Casein supramolecules and other proteins after mixing milk at 20°C 1:1 with ethanol (left) and after heating the mixture to 70°C (right) and imaged as in Fig. 6.2. Reprinted from Oommen (2004) cal laser-scattering microscopy, observed that hols. The loss of internal structure and compact- ethanol-modified casein supramolecules were ness of the small aggregates imply rearrangement smaller than native casein micelles. The presence of of individual monomeric caseins from their ini- interconnected particles or fewer large aggregates tial-interlocked structure within the casein. that are in the process of dissociation might account for these differences. As seen in Fig. 6.14, 6.10 Conclusion the dissociated casein supramolecules appear to remain interconnected and have a large amount The supramolecular structure of casein micelles of void volume that can contribute to greater can be modeled as an interlocked lattice in which hydrodynamic radius while maintaining low both casein-calcium phosphate aggregates and molecular weight and consequently low casein polymer chains act together to maintain sedimentation. casein micelle integrity. This model suggests that stabilization of calcium phosphate nanoclusters If a milk-ethanol mixture is heated, the casein by phosphoserine domains of as1-, as2-, and/or supramolecules further dissociate (Zadow 1993; b-casein would orient their hydrophobic domains O’Connell et al., 2001a) into smaller compact outward allowing interaction and binding to other particles and form aggregates (Fig. 6.14). Unlike casein molecules. Other interactions between the independent particles, they were interconnected caseins, such as calcium bridging, could also and could be considered as part of a larger aggre- occur and further stabilize the supramolecule. gate. The nuclear magnetic spectra of both urea The combination of having an interlocked lattice (6 mol/L)-treated milk and milk heated to 70°C structure and multiple interactions results in an in the presence of ethanol have been shown to be open sponge-like colloidal supramolecule that is similar (O’Connell et al., 2001b). Urea dissoci- resistant to spatial changes and disintegration ates casein supramolecules by enhancing protein unless the chemical environment is changed. The solubility and by inhibiting hydrophobic bond- occluded spaces within the supramolecule matrix ing, and presumably ethanol has a similar effect. structure would be occupied by the serum phase The increase in repulsive forces between caseins of milk comprising water along with dissolved and solvent quality may result in dissociation of lactose, ions, and other soluble substances. casein supramolecules when heated in presence Having open channels throughout the supramol- of ethanol. Horne and Davidson (1987) attributed ecule means that virtually every casein molecule this dissociation of casein supramolecules to high helix development in caseins in presence of alco-

206 D.J. McMahon and B.S. Oommen is exposed to water molecules and their confor- Aoki, T., Yamada, N., Kako, Y. and Imamura, T. (1988). mational shape will be influenced by the com- Dissociation during dialysis of casein aggregates bined interactions with other protein, with cross-linked by colloidal calcium phosphate in bovine calcium phosphate, with the water, and with casein micelles. J. Dairy Res. 55, 189–195. related entropy considerations. A distinguishing feature of this interlocked lattice model is that Bloomfield, V.A. (1979). Association of proteins. J. Dairy any change in temperature or chemical environ- Res. 46, 241–252. ment will exert a global change throughout the supramolecule. For example, the observed Bloomfield, V.A. and Mead, R.J., Jr. (1975). Structure and decrease in particle size observed when alcohol is stability of casein micelles. J. Dairy Sci. 58, added to milk can be better explained by a global 592–601. contraction of the supramolecule lattice structure rather than just collapse of a surface hairy layer. Bloomfield, V.A. and Morr, C.V. (1973). Structure of In this sense, the casein micelle itself can be con- casein micelles: physical methods. Neth. Milk Dairy J. sidered rheomorphic as well as the individual 27, 103–120. casein molecules. Bylund, G. et al. (2003). Dairy Processing Handbook, Hydrophobic interactions between caseins 2nd edn., Tetra Pak Processing Systems AB, Lund, surrounding a calcium phosphate nanocluster Sweden. would prevent complete dissociation of casein micelles when the calcium phosphate nanoclus- Corredig, M. and Dalgleish, D.G. (1996). The binding of ters are solubilized. Likewise, calcium bridging a-lactalbumin and b-lactoglobulin to casein micelles and other electrostatic interactions between in milk treated by different heating systems. caseins would prevent dissociation of the casein Milchwissenschaft 51, 123–127. micelles into casein-calcium phosphate nano- cluster aggregates when milk is cooled, or urea is Dalgleish, D.G. (2011). On the structural models of bovine added to milk, and hydrophobic interactions are casein micelles–review and possible improvements. reduced. The appearance of both polymer chains Soft Matter 7, 2265–2272. and small aggregate particles during milk synthe- sis would also be expected based on this inter- Dalgleish, D.G. and Law, A.J.R. (1988). pH-induced dis- locked lattice model of casein micelles. sociation of bovine casein micelles. I. Analysis of lib- erated caseins. J. Dairy Res. 55, 529–538. References Dalgleish, D.G. and Parker, T. G. (1979). Quantitation of Anema, S.G. and Klostermeyer, H. (1997). Heat-induced, aS1-casein aggregation by the use of polyfunctional pH-dependent dissociation of casein micelles on heat- models. J. Dairy Res. 46, 259–263. ing reconstituted skim milk at temperatures below 100°C. J. Agric. Food Chem. 45, 1108–1115. Dalgleish, D.G., Spagnuolo, P.A. and Goff, H.D. (2004). A possible structure of the casein micelle based on Anema, S.G. and Li, Y. (2003). Effect of pH on the asso- high-resolution scanning electron microscopy. Int. ciation of denatured whey proteins with casein micelles Dairy J. 14, 1025–1031. in heated reconstituted skim milk. J. Agric. Food Chem. 51, 1640–1646. Davies, F.L., Shankar, P.A. and Brooker, B.E. (1977). A heat-induced change in the ultrastructure of milk and Aoki, T., Kako, Y. and Imamura, T. (1986). Separation of its effect on gel formation in yoghurt. J. Dairy Res. 45, casein aggregates cross-linked by colloidal calcium 53–58. phosphate from bovine casein micelles by high perfor- mance gel chromatography in the presence of urea. de Kruif, C.G. (1998). Supra-aggregates of casein micelles J. Dairy Res. 53, 53–59. as a prelude to coagulation. J. Dairy Sci. 81, 3019–3028. Aoki, T., Umeda, T. and Kako, Y. (1992). The least number of phosphate groups for crosslinking of casein by col- de Kruif, C.G. and Holt, C. (2003). Casein micelle struc- loidal calcium phosphate. J. Dairy Sci. 75, 971–975. ture, functions, and interactions, in, Advanced Dairy Chemistry Proteins, 3rd edn., Vol. 1A, P.F. Fox and P.L.H. McSweeney, eds., Kluwer Academic/Plenum Publishers, New York. pp. 233–270. Dickinson, E. and Matsumura, Y. (1991). Time-dependent polymerization of b-lactoglobulin through disulfide bonds at the oil-water interface in emulsions. Int. J. Biol. Macromolecules 13, 26–30. Dosako, S., Kaminogawa, S., Taneya, S. and Yanauchi, K. (1980). Hydrophobic surface areas and net charge of as1-, k-casein and as1-casein:k -casein complex. J. Dairy Res. 47, 123–137. Euston, S.R. and Horne, D.S. (2005). Simulating the self- association of caseins. Food Hydrocolloids 19, 379–386. Farrell, H.M., Jr. (1973). Models for casein micelle for- mation. J. Dairy Sci. 56, 1195–1206. Farrell, H.M., Jr., Brown, E.M., Hoagland, P.D. and Malin, E. L. (2003). Higher order structures of caseins: a paradox, in, Advanced Dairy Chemistry Proteins, 3rd

6 Casein Micelle Structure, Functions, and Interactions 207 edn., Vol. 1A, P.F. Fox and P.L.H. McSweeney, eds., Holt, C., Wahlgren, N.M. and Drakenberg, T. (1996). Kluwer Academic/Plenum Publishers, New York. pp. Ability of a b-CN phosphopeptide to modulate the 203–232. precipitation of calcium phosphate by forming amor- Farrer, D. and Lips, A. (1999). On the self-assembly of phous dicalcium phosphate nanoclusters. Biochem. J. sodium caseinate. Int. Dairy J. 9, 281–286. 314, 1035–1039. Farrell, H.M., Jr., Malin, E.L., Brown, E.M. and Qi, P.X. (2006a). Casein micelle structure: what can be learned Holt, C., Timmis, P.A., Errington, N. and Leaver, J. from milk synthesis and structural biology? Curr. (1998). A core-shell model of calcium phosphate nan- Opin. Colloid Interface Sci. 11, 135–147. oclusters stabilized by b− casein phosphopeptides, Farrell, H.M., Jr., Qi, P.X. and Uversky, V.N. (2006b). New derived from sedimentation equilibrium and small- views of protein structure: applications to the caseins: angle X-ray and neutron-scattering measurements. protein structure and functionality, in, Advances in Eur. J. Biochem. 252, 73–78. Biopolymers: Molecules, Clusters, Networks, and Interactions. M.L. Fishman, P.X. Qi, and L. Wisker, Horne, D.S. (1986). Steric stabilization and casein micelle eds., Am. Chem. Soc., Washington. pp. 52–70. stability. J. Coll. Interf. Sci. 111, 250–260. Garnier, J. (1973). Models of casein micelle structure. Neth. Milk Dairy J. 27, 240–248. Horne, D.S. (1992) Alcohol stability, in, Advanced Dairy Garnier, J. and Ribadeau-Dumas, B. (1970). Structure of Chemistry Proteins, 2nd edn., Vol. 1, P.F. Fox, ed., the casein micelle: a proposed model. J. Dairy Res. Elsevier Applied Science, New York. pp. 657–589. 37,493–504. Garnier, J., Yon, J. and Mocquot, G. (1964). Contribution Horne, D.S. (1998). Casein interactions: casting light on to the study of the association between k- and as- the black boxes, the structure of dairy products. Int. caseins at neutral pH. Biochimica et Biophyica. Acta Dairy J. 8, 171–177. 82, 481–493. Gebhardt, R., Takeda, N., Kulozik, U. and Doster, W. Horne, D.S. (2002a). Casein structure, self assembly and (2011). Structure and stabilizing interactions of casein gelation. Curr. Opin. Coll. Interf. Sci. 7, 456–461. micelles probed by high-pressure light scattering and FTIR. J. Phys. Chem. B. 115, 349–2359. Horne, D.S. (2002b). Caseins, micellar structure, in, Heertje, I., Visser, J. and Smits, P. (1985). Structure forma- Encyclopaedia of Dairy Sciences, Vol. 3, R. Roginski, tion in acid milk gels. Food Microstruct. 4, 267–278. P.F. Fox and J.W Fuquay, eds., Academic Press, NY. Helminen, H.J. and Ericsson, J.L.E. (1968). Studies on pp. 1902–1909. mammary gland involution. 1. On the ultrastructure of the lactating mammary gland. J. Ultrastruct. Res. 25, Horne, D.S. (2006). Casein micelle structure: models and 193–213 muddles. Curr. Opin. Coll. Interf. Sci. 11, 148–153. Hojou, K., Oikawa, T., Kanaya, K., Kimura, T., and Adachi, K. (1977). Some applications of ion beam Horne, D.S. and Davidson, C.M. (1987). Alcohol stability sputtering to high resolution electron microscopy. of bovine skim-milk. Anomalous effects with Micron 8, 151–170. trifluoroethanol. Milchwissenschaft 42, 509–512. Holt, C. (1992). Structure and stability of bovine casein micelles. Adv. Prot. Chem. 43, 63–151. Horne, D.S., Parker, T.G. and Dalgleish, D.G. (1988). Holt, C. (1998). Casein micelle substructure and calcium Casein micelles, polycondensation, and fractals, in, phosphate interactions studied by sephacryl column Food Colloids, R.D. Bee, P. Richmond and J. Mingins, chromatography. J. Dairy Sci. 81, 2994–3003. eds, Royal Soc. Chem., London. pp. 400–406. Holt, C. and Horne, D.S. (1996). The hairy casein micelle: evolution of the concept and its implications for dairy Kalab, M., Emmons, D.B., and Sargant, A.G. (1976). technology. Neth. Milk Dairy J. 50, 85–111. Milk gel structure: V. Microstructure of yoghurt as Holt, C. and Sawyer, L. (1993). Caseins as rheomorphic related to the heating of milk. Milchwissenschaft 31, proteins: interpretation of the primary and secondary 402–408. structures of the as1-, b-, and k-caseins. J. Chem. Soc. Faraday Trans. 89, 2683–2692. Karlsson, A., Ipsen, R. and Ardö, Y. (2007). Observations Holt, C., Davies, D.T. and Law, A.J.R. (1986). Effects of casein micelles in skim milk concentrate by trans- of colloidal calcium phosphate content and free cal- mission electron microscopy. LWT-Food Sci. Technol. cium ion concentration in the milk serum on the dis- 40, 1102–1107. sociation of bovine casein micelles. J. Dairy Res. 53, 557–572. Kim, B.Y. and Kinsella, J.E. (1989). Effect of temperature Holt, C., de Kruif, C.G., Tunier, R. and Timmons, P.A. and pH on the coagulation of casein. Milchwissenschaft (2003). Substructure of bovine casein micelles by 44, 622–625. small angle X-ray and neutron scattering. Colloids Surf. A 213, 275–284. Kirchmeuer, O. (1973). Arrangement of components, electrical charge and interaction energies of casein micelles. Neth. Milk Dairy J. 27, 191–198. Knoop, A.M., Knoop, E. and Wiechen, A. (1979). Sub- structure of synthetic casein micelles. J. Dairy Res. 46, 357–350. Kumosinski, T.P., Pessen, H., Farrell, H.M., Jr. and Brumberger, H. (1988). Determination of the quater- nary structure of bovine caseins by small-angle X-ray scattering. Arch. Biochem. Biophys. 266, 548–561. Lin, S.H.C., Leong, L.S., Dewan, R.K., Bloomfield, V.A. and Morr, C.V. (1972). Effect of calcium ion on the structure of native bovine casein micelles. Biochem. 11, 1818–1821.

208 D.J. McMahon and B.S. Oommen Linderstrøm Lang, K. (1929). Studies on casein. III. On Pierre, A. and Brule, G. (1981). Mineral and protein equi- the fractionation of casein. Compt. Rend. Trav. Lab. libria between the colloidal and soluble phases of milk Carlsberg. 36(9), 1. at low temperature. J. Dairy Res. 48, 417–428. Lucey, J. A. (2002). Formation and physical properties of Pierre, A., Brule, G. and Fauquant, J. (1983). Etude de la milk protein gels. J. Dairy Sci. 85, 281–294. mobilite du calcium dans le lait a l’aide du calcium. Lait. 63, 473–489. Malin, E.L., Brown, E.M., Wickham, E.D. and Farrell, H.M., Jr. (2005). Contributions of terminal peptides to Pignon, F., Belina, G., Narayanan, T., Paubel, X., Magnin, the associative behavior of as1-casein. J. Dairy Sci. A. and Gésan-Guiziou, G. (2004). Structure and rheo- 88, 2318–2328. logical behavior of casein micelle suspensions during ultrafiltration process. J. Chem. Phys. A 121, Marchin, S., Putaux, J.-L., Pignon, F. and Léonil, J. 8138–8146. (2007). Effects of the environmental factors on the casein micelle structure studied by cryo transmission Rasmussen, L.K., Johnsen, L.B., Tsiora, A., Sørenson, electron microscopy and small-angle X-ray scattering/ E.S., Thomsen, J.K., Nielsen, N.C., Jakobsen, H.J. and ultrasmall-angle X-ray scattering. J. Chem. Phys. Petersen, T.E. (1999). Disulphide-linked caseins and 126(4), 045101 (1–10). casein micelles. Int. Dairy J. 9, 215–218. McMahon, D.J. and Brown, R.J. (1984). Composition, Roefs, S.P.F.M., Walstra, P., Dalgleish, D.G. and Horne, structure, and integrity of casein micelles: a review. D.S. (1985). Preliminary note on the change in casein J. Dairy Sci. 67, 499–512. micelles caused by acidification. Neth. Milk Dairy J. 39, 119–122. McMahon, D.J. and McManus, W.R. (1998). Rethinking casein micelle structure using electron microscopy. Rollema, H.S. (1992). Chemistry of milk proteins, in, J. Dairy Sci. 81, 2985–2993. Developments in Dairy Chemistry Proteins, Vol. 1, P.F. Fox, ed., Applied Science, London. pp. 111–140. McMahon, D.J. and Oommen, B.S. (2008). Supramolecular structure of the casein micelle. J. Dairy Sci. 91, 1709–1721. Rollema, H.S. and Brinkhuis, J.A. (1989). A 1H-NMR study of bovine casein micelles; influence of pH, McMahon, D.J., Du, H., McManus, W.R. and Larsen, temperature and calcium ions on micellar structure. K.M. (2009). Microstructural changes in casein supra- J. Dairy Res. 56, 417–425. molecules during acidification of skim milk. J. Dairy Sci. 92, 5854–5867. Rose, D. (1969). A proposed model of micelle structure in bovine milk. Dairy Sci. Abstr. 31, 171–175. Meyer, J.L. and Angino, E.E. (1977). The role of trace met- als in calcium urolithiasis. Invest. Urol. 14, 347–350. Schmidt, D.G. (1980). Colloidal aspects of casein. Neth. Milk Dairy J. 34, 42–64. Morr, C.V. (1967). Effect of oxalate and urea upon ultra- centrifugation properties of raw and heated skim milk Schmidt, D.G. and Payens, T.A.J. (1976). Micellar aspects casein micelles. J. Dairy Sci. 50, 1744–1751. of casein, in, Surface and Colloid Science, Vol. 9, E. Matijevic, ed., Wiley-Interscience, New York. pp. O’Connell, J.E., Kelly, A.L., Auty, M.A.E., Fox, P.F. and 165–229. de Kruif, C.G. (2001a). Ethanol-dependent tempera- ture-induced dissociation of casein micelles. J. Agric. Schmidt, D.G., Koops, J. and Westerbeek, D. (1977). Food Chem. 49, 4420–4423. Properties of artificial casein micelles. 1). Preparation, size distribution and composition. Neth. Milk Dairy J. O’Connell, J.E., Kelly, A.L., Fox, P.F. and de Kruif, C.G. 31, 328–341. (2001b). Mechanism for the ethanol-dependent heat- induced dissociation of casein micelles. J. Agric. Food Schmidt, D.G., van der Spek, C.A., Buchheim, W. and Chem. 49, 4424–4428. Hinz, A. (1974). On the formation of artificial casein micelles. Milchwissenschaft 29, 455–459. Oommen, B.S. (2004). Casein Supramolecules: Structure and Coagulation Properties, Ph.D. Dissertation, Utah Schmidt, D.G., Walstra, P. and Buchheim, W. (1973). The State University, Logan, UT, USA. size distribution of casein micelles in cow’s milk. Neth. Milk Dairy J. 27, 128. Payens, T.A.J. (1966). Association of caseins and their possible relation to structure of the casein micelle. Singh, H., Roberts, M.S., Munro, R.A. and Teo, C.T. J. Dairy Sci. 49, 1317–1324. (1996a). Acid-induced dissociation of casein micelles in milk: effects of heat treatment. J. Dairy Sci. 79, Payens, T.A.J. (1968). Self association and complex forma- 1340–1346. tion of as1- and b-caseins. Biochemical J. 108, 14p. Singh, H., Sharma, R., Taylor, M.W. and Creamer, L.K. Payens, T.A.J. and Markwijk, V. (1963). Some features of (1996b). Heat-induced aggregation and dissociation of the association of b-casein. Biochimi. Biophys. Acta protein and fat particles in recombined milk. Neth. 71, 517–530. Milk Dairy J. 50, 149–166. Payens, T.A.J. and Vreeman, H.J. (1982). Casein micelles Slattery, C.W. (1976). Review: casein micelle structure; and micelles of k- and b-casein, in, Solution Behavior an examination of models. J. Dairy Sci. 59, of Surfactants: Theoretical and Applied Aspects, K.L. 1547–1556. Mittal and E.J. Fendler, eds., Vol. 1, Perseus Publishing, Cambridge, MA. pp. 543–571. Slattery, C.W. (1979). A phosphate-induced sub-micelle equilibrium in reconstituted casein micelle systems. Pessen, H., Kumosinski, T.F. and Farrell, H.M., Jr. (1989). J. Dairy Res. 46, 253–258. Small-angle x-ray scattering investigation of the micellar and submicellar forms of bovine casein. Slattery, C.W. and Evard, R. (1973). A model for the for- J. Dairy Res. 56, 443–451. mation and structure of casein micelles from subunits

6 Casein Micelle Structure, Functions, and Interactions 209 of variable composition. Biochim. Biophys. Acta. 317, Tuiner, R. and de Kruif, C.G. (2002). Stability of casein 529–538. micelles in milk. J. Chem. Phys. 117, 1290–1295. Smith, E., Clegg, R. and Holt, C. (2004). A biological perspective on the structure and function of caseins Udabage, P., McKinnon, I.R. and Augustin, M.A. (2003). and casein micelles. Int. J. Dairy Technol. 57, The use of sedimentation field flow fractionation and 121–126. photon correlation spectroscopy in the characteriza- Snøeren, T.H.M., Klok, H.J., van Hooydonk, A.C.M. and tion of casein micelles. J. Dairy Res. 70, 453–459 Dammam, A.J. (1984). The voluminosity of casein micelles. Milchwissenschaft 39, 461–463. van Hooydonk, A.C.M., Boerrigter, I.J. and Hagedoorn, Swaisgood, H.E. (1992). Chemistry of the caseins, in, H.G. (1986). pH-induced physico-chemical changes Advanced Dairy Chemistry Proteins, 3rd edn., Vol. of casein micelles in milk and their effect on rennet- 1A, P.F. Fox and P.L.H. McSweeney, eds., Kluwer ing: 2. Effect of pH on renneting of milk. Neth. Milk Academic/Plenum Publishers, New York. pp. 63–110. Dairy J. 40, 297–313. Swaisgood, H.E. (2003). Chemistry of the caseins, in, Advanced Dairy Chemistry Proteins, Vol 1A, P.F. Fox Walstra, P. (1990). On casein micelles. J. Dairy Sci. 73, and P.L.H. McSweeney, eds., Kluwer Academic/ 1965–1979. Plenum Publishers, New York. pp. 139–187. Swaisgood, H.E. and Brunner, J.R. (1973). The caseins. Walstra, P. (1999). Casein sub-micelles: do they exist? Int. CRC Crit. Rev. Food Technol. 3, 375–414. Dairy. J. 9, 189–192. Thompson, M.P., and Farrell, H.M., Jr. (1973). The casein micelle-the forces contributing to its integrity. Neth. Waugh, D.F. and Noble, R.W. (1965). Casein micelles: Milk Dairy J. 27, 220–239. formation and structure. J. Am. Chem. Soc. 78, Thurn, A., Buchard, W. and Niki R. (1987). Structure of 2246–2257. casein micelles 2. as1-. Coll. Poylmer Sci. 265, 653–666. Trejo, R., Dokland, T., Jurat-Fuentes, J. and Harte, F. Yoshikawa, M., Sasaki, R. and Chiba, H. (1981). Effects (2011). Cryo-transmission electron tomography of of chemical phosphorylation of bovine casein compo- native milk casein micelles. J. Dairy Sci. 94, nents on the properties related to casein micelle forma- 5770–5775. tion. Agric. Biol. Chem. 45, 909–914. Zadow, J.G. (1993). Alcohol-mediated temperature- induced reversible dissociation of the casein micelle in milk. Aust. J. Dairy Technol. 48, 78–81. Zhang, Z.P., Fujii, M. and Aoki, T. (1996). Behavior of calcium and phosphate in artificial casein micelles. J. Dairy Sci. 79, 1722–1727.

b-Lactoglobulin 7 L. Sawyer 7.1 Introduction versions (Pervaiz and Brew, 1985; Sawyer, 1987; North, 1991; Flower, 1996). The structurally The lipocalin family to which b-lactoglobulin, conserved regions are highlighted in the lacto- b-Lg, belongs has been expanding rapidly over globulin sequence comparison shown in a later the past couple of decades and now comprises at section. North (1989, 1991) noticed that the con- least 40 examples, widely spread throughout the served sequences are grouped at the base of the biosphere (Åkerström et al., 2006; Grzyb et al., calyx furthest from its entrance such that a recep- 2006), probably indicative of a bacterial origin tor recognition function is implied—a transporter (Sanchez et al., 2006). The family has low needs to recognise its destination and might also sequence identity, generally less than 25%, but need to signal whether it is carrying a ligand. The have a well-conserved tertiary structure comprising relatedness of the lipocalins is further supported an antiparallel b-barrel or calyx. Mostly, members by the similarity in gene sequences (Ali and have a subunit molecular weight of 18–20 kDa, Clark, 1988; Salier, 2000; Simpson and Nicholas, but several domains of larger proteins have also 2002; Sanchez et al., 2006). The function of b-Lg been found to adopt the lipocalin fold, some have in relationship to its being a lipocalin will be dis- enzymic activity and several, like insecticyanin cussed after considering its molecular properties. and crustacyanin, bind chromophores (Table 7.1). However, the functions of quite a number are In the b-Lg chapter in the third edition of still, at best, ill-defined. Many seem to bind and Advanced Protein Chemistry (Sawyer, 2003), the transport a hydrophobic or labile small molecule, review covered the literature to the end of 2000. In and some members appear to have been identified the decade that has followed, there has been a principally as allergens, including Bos d 2 which, significant body of work on the protein, further although a bovine lipocalin, is quite distinct from defining its structure, properties and increasingly, b-Lg (Rouvinen et al., 1999). its applications in the general area of food and nutrition. Roughly one paper a day has been added A protein sequence signature for the lipocalins covering both pure (i.e. properties and behaviour is provided in the PRINTS database (Attwood of b-Lg in its own right) and applied aspects, and et al., 2003) and is an improvement on earlier it is the aim of this chapter to deal mainly with the molecular properties of the protein. The more L. Sawyer (*) applied aspects are covered elsewhere (e.g. School of Biological Sciences, The University of Thompson et al., 2009; see also Volume 1B). Edinburgh, King’s Buildings, Mayfield Road, Edinburgh EH9 3JR, UK b-Lg is the major whey protein secreted in the e-mail: [email protected] milk of ruminants like the cow or sheep. It is also found in the milk of monogastrics like the pig, P.L.H. McSweeney and P.F. Fox (eds.), Advanced Dairy Chemistry: Volume 1A: Proteins: Basic Aspects, 211 4th Edition, DOI 10.1007/978-1-4614-4714-6_7, © Springer Science+Business Media New York 2013

Table 7.1 Selected members of the lipocalin family showing their wide distributiona 212 L. Sawyer Protein Sourceb Amino Locationb Ligandc Function PDBd References b-Lactoglobulin, Bos d 5 Many mammals acids Milk Fatty acids, Transport/ entry Brownlow et al. (1997), allergen 162 vitamins A, D? transfer? Hoedemaeker et al. (2002) Glycodelin Human, baboon Amniotic fluid Retinol Differentiation? 1BEB, Seppala et al. (2009) Retinol-binding proteine Mammals, chicken 162 Blood serum Retinol Transport 1EXS Cowan et al. (1990) Apolipoprotein D Human 183 Serum, gross breast Progesterone Acute phase Eichinger et al. (2007) 169 cystic disease protein – Complement protein C8g Human Serum Part of membrane Immune system 1JYD 182 attack complex 2HZQ a1-Acid glycoprotein, Human Serum Acute phase orosomucoid 192 Saccharides protein 1LF7 Ortlund et al. (2002) Neutrophil gelatinase- Human Serum, uterine Anti-bacterial, associated lipocalin, LCN2, 179 secretion Fatty acids? acute phase 3KQ0 Schonfeld et al. (2008) siderocalin Human protein Tear lipocalin, von Ebner’s 162 Tears, saliva Odorants Transport? 1DFV Holmes et al. (2005) gland protein Cow, pig 2-sec-Butyl-4,5- Odorant-binding protein Mouse 159 Nasal mucous dihydrothiazole Transduction? 1XKI Breustedt et al. (2005) Major urinary protein (MUP) 162 Urine Prostaglandin H2 Marking? Rat, human 1DZK Vincent et al. (2001) Prostaglandin D synthase 168 Brain, cerebrospinal Astaxanthin PGD2 synthesis 3KFF Perez-Miller et al. (2010) Cow fluid Histamine/NO Bos d 2 lipocalin allergen Lobster 156 Sweat Biliverdin IXg Camouflage? 2CZT, Kumasaka et al. (2009), a-Crustacyanin, C1 subunit Tick 181 Carapace Violaxanthin Vasodilation 3O22 Zhou et al. (Unpublished) Nitrophorin Butterfly 170 Saliva Camouflage 1BJ7 Rouvinen et al. (1999) Bilin-binding protein Arabidopsis 173 Epidermis Vaccenic acid Zeaxanthin 1I4U Gordon et al. (2001) Domain of violaxanthin thaliana 185 Thylakoid lumen synthesis 1NP1 Weichsel et al. (1998) de-epoxidase Bacteria Stress response 1BBP Huber et al. (1987) Bacterial lipocalin, BLC 168 Membrane 3CQN Arnoux et al. (2009) 3MBT Schiefner et al. (2010) The PRINTS lipocalin signature (Attwood et al., 2003) identifies three structurally conserved regions beginning at residues 13, 94 and 121 in the b-Lg numbering aA special issue of Biochimica et Biophysica Acta, 1482(2) (2000) is devoted to the lipocalins. See also Åkerström et al. (2006) bSource and location from which the protein can be derived. The distribution is generally more widespread cMany lipocalins bind a variety of ligands, and often the physiological ligand is unknown dCode for the coordinate set in the Protein Data Bank: http://www.ebi.ac.uk eThe cellular RBP, like the related fatty acid-binding protein, is a distinct protein with a 10-stranded barrel made up of fewer amino acids belonging to the wider calycin family

7 b-Lactoglobulin 213 horse, dog, cat and in marsupials, but it is absent Fig. 7.1 The signal peptides of several b-lactoglobulins from the milk of humans, lagomorphs and (b-Lg) showing the high degree of conservation across the rodents. Bovine b-Lg can be isolated readily, and widely diverse species. Identities are shown as asterisks, since its isolation from milk by Palmer (1934), it near similarity as colon and more distant similarity as has been, and still is, used extensively as a conve- period nient small protein on which to try out new tech- niques, both experimental and theoretical. As from about mid-pregnancy (Simpson and well as Sawyer (2003), a number of specific Nicholas, 2002; see Chap. 14). The b-Lg gene reviews on the properties of b-Lg have been pub- comprises seven exons, the last of which is not lished over the years (e.g. Tilley, 1960; Townend translated in the mature protein. mRNA coding et al., 1969; McKenzie, 1971; Hambling et al., for b-Lg which is specific to the mammary tis- 1992; Qin et al., 1998b; Kontopidis et al., 2004), sue is translated to yield a 180 amino acid pre- together with several others on milk proteins in b-Lg, whose signal peptides, some of which are general, which contain substantial sections on the shown in Fig. 7.1, contain highly conserved, properties of the protein (e.g. McKenzie, 1967; largely hydrophobic amino acids (The Uniprot Lyster, 1972; Thompson and Farrell, 1974; Consortium, 2008). Jenness, 1979, 1985; Swaisgood, 1982; Kinsella and Whitehead, 1989; Fox, 1995; Farrell et al., After removal of the signal peptide (Fig. 7.1), 2004; Edwards et al., 2009). the mature protein undergoes disulphide bridge formation, within the rough endoplasmic reticu- Binding studies carried out on the bovine pro- lum. Transport to the Golgi and incorporation tein in vitro have shown that it can bind a variety into secretory vesicles precede secretion into the of ligands, most of which are small, hydrophobic lumen, where b-Lg accumulates in the milk molecules like fatty acids or retinol. b-Lg under- before removal by suckling. goes several conformational changes between pH 2 and pH 9, possibly the most important of which, The promoter region of the b-Lg gene not only the N ↔ R, or Tanford, transition, occurs in the directs expression specifically to the mammary physiological pH range. The ruminant protein is gland but also promotes high levels of expres- a dimer under physiological conditions, whereas sion. Consequently, transgenic mice have the b-Lg from other species appears to be mono- expressed in their milk native (Simons et al., meric. However, now that the crystal structures of 1987) or modified (McClenaghan et al., 1999) b- b-Lg at several pH values have become available, Lg from sheep, goat (Ibanez et al., 1997) and cow a detailed molecular explanation of most of the (Bawden et al., 1994; Hyttinen et al., 1998). solution properties is available (Qin et al., 1998a; Further, studies on the promoter region have Sakurai et al., 2009). identified the minimum necessary for expression (Whitelaw et al., 1992) as well as binding sites This chapter will expand upon the above out- for, and the differing effects of, various promot- line to discuss the structure, molecular properties ers (e.g. Rosen et al., 1999; Pena and Whitelaw, and possible function of the protein in some 2005; Braunschweig, 2007; Kotresh et al., 2009; detail, covering the salient literature from the past Fraser et al., 2009). eight decades. Inevitably, it can provide only an overview, coloured by the individual prejudices of the author. 7.2 Biosynthesis and Secretion Major lactoproteins, including b-Lg, are biosyn- thesised within the secretory epithelial cells of the mammary gland under endocrine control

214 L. Sawyer Developments in mammary cell lines (German has also been reported. Site-directed mutagenesis and Barash, 2002), PCR-RFLP (e.g. Caroli et al., of recombinant protein provides a powerful 2009), microarray technology (Chessa et al., method for modifying the properties, which, for 2007) and of course genome sequences (Hubbard milk proteins, is providing a route to improved et al., 2009; Lemay et al., 2009; Elsik et al., functionalities (Batt et al., 1994; Whitelaw, 1999; 2009) have led to more detailed studies of milk see Chap. 16). biosynthesis in general and that of b-Lg in par- ticular, not only in bovine, ovine and caprine spe- 7.3 Distribution cies but also in horse (Uniacke-Lowe et al., 2010), donkey (Guo et al., 2007) and the less The composition of milk varies with time since economically important marsupial (Joss et al., parturition, with species and also with season, the 2009) and fur seal (Cane et al., 2005) milks. For last presumably related to dietary habit. example, Reichenstein et al. (2005) investigated Qualitative and quantitative methods for the sep- a novel regulatory element in the ovine b-Lg pro- aration of whey proteins, useful for detecting the moter using a b-Lg-luciferase construct. The presence of b-Lg, have been reported by, inter ability to detect polymorphisms efficiently has alia, Davies (1974), Strange et al. (1992) and revolutionised the study of milk production trait Otte et al. (1994). However, neither electropho- loci (e.g. Caroli et al., 2009) and the comparative retic mobility nor polyclonal antibody cross-reac- biology of milk proteins (Simpson and Nicholas, tivity alone should be taken as proof of the 2002; Lemay et al., 2009; Elsik et al., 2009). presence of b-Lg (Bertino et al., 1996; Conti Such studies have highlighted a number of non- et al., 2000). During the past two decades, restric- coding mutations within the b-Lg gene that tion fragment length polymorphism (Lien et al., appear to be responsible for varying levels of pro- 1990), and the polymerase chain reaction (PCR) tein expression (Ganai et al., 2009) which in turn have been used to investigate the distribution of affect the commercial aspects of the milk b-Lg (e.g. Jadot et al., 1992; Prinzenberg and production. Erhardt, 1999). Isoelectric focussing, 2D and capillary electrophoresis have also been used suc- Although bovine and ovine b-Lgs were over- cessfully (Paterson et al., 1995; Veledo et al., expressed in Escherichia coli, Lactobacillus 2005), more recently supplemented by mass casei, Saccharomyces cerevisiae and spectrometry and microarray technologies Kluyveromyces lactis as the near-native protein (Chessa et al., 2007). (Batt et al., 1990; Rocha et al., 1996; Hazebrouck et al., 2007) or as a fusion protein (Ariyaratne Since the initial preparation of b-Lg from et al., 2002), over-expression at levels in excess bovine (Bos taurus) milk (Palmer, 1934), dimeric of 100 mg b-Lg/L of culture supernatant was b-Lg has been isolated from the milk of a number achieved only in Pichia pastoris (Kim et al., of other ruminants, and monomeric b-Lg has 1997). Recently, however, Invernizzi et al. (2008) been purified from the milk of several nonrumi- have achieved expression levels around 100 mg/L nant livestock species (Table 7.2). b-Lg has also of soluble, secreted b-Lg in E. coli, but mutation been detected in milk of other species, but the of Cys121 led to insolubility. Ponniah et al. (2010), state of its association in these cases is uncertain. on the other hand, have reported overproduction For instance, although McKenzie et al. (1983) of soluble bovine b-Lgs A and B in E. coli using suggest that kangaroo b-Lg has a monomeric a coding sequence optimised for the bacterium structure, later electrophoretic evidence implies and co-expressing a disulphide isomerase. This that wallaby b-Lg exists as monomers, dimers system seems capable of producing mutated and tetramers (Woodlee et al., 1993). forms of the protein in quantities around 10 mg/L, labelled if required for NMR studies. Heterologous While the milk of ruminants contains the b-Lg expression of both porcine (Invernizzi et al., from a single gene, which may exist in distinct 2004) and equine (Kobayashi et al., 2000) b-Lgs allelic forms, the milk from dog, dolphin, cat,

7 b-Lactoglobulin 215 Table 7.2 Distribution of b-lactoglobulin in the milk of various species AB C D E +a Dib,c 1.8–5.0 Cow (Bos taurus, B. javanicus, B. grunniens, B. indicus) +a ?Di +a ?Di* 1.4 Buffalo (Bubalus arnee, B. bubalis) +a ?Di* 2.8 +a ?Di* 2.8–3.0 Bison (Bison bison) + ?Di + Did,e 0.6 Musk ox (Ovibos moschatus) +a ?Di* 2.3 +a ?Di* 16.2 Eland (Taurotragus oryx) +a Di 14.1 +a ?Di* 10.1 Goat (Capra hircus) +a ?Di* +a ?Di* <0.1118 Sheep (Ovis aries, O. ammon musimon) +a ?Di* +a ?Di* 0 Red deer (Cervus elaphus L.) +a ?Di* 0 Nm ?Monof 0 European elk (Alces alces L.) Nm ?Monog,h Reindeer (Rangifer tarandus L.) −*a Mono ±i Mono White-tailed deer (Odocoileus virginianus) ±i,j Monok +a Mono* Fallow deer (Dama dama) Nm Mono*l Nm Monom Caribou (Rangifer arcticus) Nm Monon Giraffe (Giraffa camelopardalis) Nm Monon Nm Monon Okapi (Okapia johnstoni) −o ?Mono* Nm Mono*p Pronghorn antelope (Antilocapra americana) Nm Monoq Nm Mono Giant panda (Ailuropoda melanoleuca) Nm Monor Nm ?Mono Bears (Ursus americanus, U. maritimus, U. arctos horribilis, U. Nm ?Mono arctos yesoensis, U. arctos middendorffi, U. malayanus) ±s None*t −a None Peccary (Pecari tajacu) ±u ?None −v Nonev Pig (Sus scrofa domestica) − Nonew Nm Nonex Horses (Equus caballus, E. quagga, E. asinus) Nm Noney Rhinoceros (Diceros bicornis) Rhinoceros (Rhinoceros unicornis) Fur seals (Callorhinus ursinus, Arctocephalus gazella, A. pusillus doriferus, A. tropicalis) Dolphin (Tursiops truncatus) Manatee (Trichechus manatus latirostris) Dog (beagle) (Canis familiaris) Cat (Felis catus) Grey kangaroo (Macropus giganteus, M. rufus, M. eugenii) Echidna (Tachyglossus aculeatus) Brush tail possum (Trichosurus vulpecula) Platypus (Ornithorhynchus anatinus) Yellow baboon (Papio hamadryas) Macaque (Macaca fascicularis) Human (Homo sapiens) Rabbit (Oryctolagus cuniculus) Camel (Camelus dromedarius) Llama (Lama glama L.) Mouse (Mus musculus) Rat (Rattus norvegicus) Guinea pig (Cavia porcellus) Column A Species Column B Presence of b-lactoglobulin , Cross-reactivity or other information , Some sequence information available (continued)

216 L. Sawyer Table 7.2 (continued) Column C Cross-reaction to anti-bovine antisera +, BLG detected by anti-bovine BLG antisera −, BLG not detected by anti-bovine BLG antisera Column D ±, BLG detected by anti-bovine BLG antisera, but only at higher titres N cross-reactivity to anti-bovine BLG antisera not measured State of association Column E Di dimeric BLG detected Mono monomeric BLG detected ?Mono, ?Di BLG present, but its state of association unknown None no BLG present ?None probably absent *Cross-reactivity to anti-bovine BLG antisera is assumed to indicate a dimeric form of the protein. In the case of human, the cross reactivity is an artefact Quantity (mg/mL) aLyster et al. (1966); bBull and Currie (1946); cBell et al. (1981a, c); dBell and McKenzie (1967b); eGodovac-Zimmer- mann et al. (1987); fHudson et al. (1984); gAndo et al. (1979); hJenness et al. (1972); iLiberatori et al. (1979a); jBell et al. (1981c); kGodovac-Zimmermann et al. (1988); lNath et al. (1993); mCane et al. (2005); nPervaiz and Brew (1986); oHalliday et al. (1991); pMcKenzie et al. (1983); qTeahan et al. (1991); rWarren et al. (2008); sLiberatori et al. (1979b); tBell and McKenzie (1964); uLiberatori et al. (1979c); vFernandez and Oliver (1988); wSimons et al. (1987); xHen- nighausen and Sippel (1982); yBrew and Campbell (1967) horse and marsupials contains the product of two, and Macaca mulatta—Azuma and Yamauchi, or in some cases three, distinct genes (Collet and 1991; Kunz and Lönnerdal, 1994) and the yellow Joseph, 1995; Piotte et al., 1998). While dogs, baboon (Papio hamadryas, Hall et al., 2001), in horses and donkeys, and cats express two or which three alleles have been identified. As more three distinct forms of b-Lg, marsupials produce complete mammalian genomes become avail- a b-Lg and a late lactation protein more closely able, a more detailed distribution will emerge, related to odorant-binding protein (Flower, 1996). perhaps allowing a clear functional assignment to Possum also produces a third lipocalin that is the protein. Of particular interest in this regard is most like the major urinary proteins from rat and that of the orangutan (Pongo spp.) whose rela- mouse (Piotte et al., 1998; Watson et al., 2007). tionship to humans is between that of the chim- b-Lg II of the cat, horse and donkey appears to be panzee and the baboon (Hubbard et al., 2009), most closely related to the b-Lg pseudogenes but in which at this stage, only the lipocalin (Piotte et al., 1998; Pena et al., 1999) identified glycodelin appears to have been identified. in cow (Passey and MacKinlay, 1995) and goat (Folch et al., 1996). 7.4 Isolation b-Lg is absent from the milk of the Camelidae The isolation of b-Lg from milk is a simple pro- (Kappeler et al., 2003; Zhang et al., 2005). b-Lg cedure, involving just four stages: removal of fat, is also absent from rodent and lagomorph milks. removal of the caseins, fractionation of the whey The lack of hybridisation between cDNA from a proteins and the final purification of b-Lg. Since rat mammary library and cDNA from sheep b-Lg each stage can be carried out in a number of (Simons et al., 1987) preceded the whole genome ways, various protocols are available and others studies of rat and mouse that confirmed the obser- continue to be published. vation (Hubbard et al., 2009). Similarly, b-Lg is absent from human and chimpanzee milk. The original isolation by Palmer (1934) was Perhaps surprisingly, however, some primates do superseded by that of Aschaffenburg and Drewry produce b-Lg: the macaque (Macaca fascicularis

7 b-Lactoglobulin 217 (1957) and scaled up by Mailliart and Ribadeau- by gel filtration, simplified in some cases by Dumas (1988). Armstrong et al. (1967) replaced expression of a fusion protein followed by affinity the potentially harsh pH treatment by precipita- chromatograph (e.g. Ariyaratne et al., 2002). tion at pH 3.5, and Monaco et al. (1987) used DEAE-cellulose chromatography to keep the pH The amount of b-Lg obtained by the various around 6.5 throughout. More recent methods, methods depends upon both the procedure used and ideally suited for bulk separations, rely on gel the quantity of b-Lg in the initial milk, which is filtration, membrane filtration and ultrafiltration known to vary with species, season and time since (e.g. Brans et al., 2004; Saufi and Fee, 2009), ion parturition. The quantity of b-Lg isolated from the exchange and hydrophobic interaction chroma- milk of a few species is given in Table 7.2. tography (e.g. Kristiansen et al., 1998; Lozano et al., 2008). Affinity chromatography and 7.5 Genetic Variants and Primary exploiting b-Lg complexes of retinol (Heddleson Structure et al., 1997) or retinal (Vyas et al., 2002) have been described also, the latter finding that a Over the past two decades, there has been fluidised bed produced the best results. significant interest in the genetic variability of Presumably because other methods work well, milk proteins with respect to production and pro- little work on ethanol fractionation of b-Lg has cessing properties, including the effect of the been reported since that of Bain and Deutsch principal b-Lg variants (Hill et al., 1996). The (1948) on cow and goat milk which suggested the effects are more thoroughly reviewed elsewhere protein had denatured. Dimethyl sulphoxide can (e.g. Caroli et al., 2009; Ganai et al., 2009). also be used although there appears to be little advantage (Arakawa et al., 2007). Although Li (1946) and Polis et al. (1950) sepa- rated two components of b-Lg, it was Aschaffenburg Other dimeric b-Lgs can be obtained by these and Drewry (1955, 1957) who showed that the two or similar methods. Thus, first reports exist for components in bovine milk were genetically deter- the isolation of b-Lg from yak (Grosclaude et al., mined. Genetic variants of b-Lg also exist in other 1976; Ochirkhuyag et al., 1998), red deer ruminant species, while distinct genes appear in (McDougall and Stewart, 1976), reindeer (Heikura other mammals (Piotte et al., 1998; Pena et al., et al., 2005), water buffalo (Kolde et al., 1981), 1999). Ion exchange was used to separate the two sheep (Maubois et al., 1965) and goat (Kalan and most common bovine genetic variants, A and B Basch, 1969). Both goat (Préaux et al., 1979) and (Piez et al., 1961), but many other genetic variants sheep (Godovac-Zimmermann et al., 1987) b-Lgs of bovine b-Lg have now been identified (Farrell have also been isolated by gel filtration (cf., et al., 2004). Other convenient phenotyping meth- Davies, 1974; Strange et al., 1992). ods include gel electrophoresis of whole milk (Davies, 1974; Lowe et al., 1995), capillary Nonruminant b-Lg can be isolated with equal electrophoresis (Paterson et al., 1995; de Frutos ease using procedures similar to those already et al., 1997; Schopen et al., 2009), isoelectric described. Pig b-Lg has been purified by Jones and focussing (Godovac-Zimmermann et al., 1990; Kalan (1971), Ugolini et al. (2001) and Kessler and Fernandez-Espla et al., 1993; Dorji et al., 2010), Brew (1970), the latter method being adapted for HPLC (Presnell et al., 1990; Miranda et al., 2004), the monomeric b-Lgs from dolphin, manatee, beagle molecular biological techniques (Jadot et al., 1992; (Pervaiz and Brew, 1986) and horse (Godovac- Schlee et al., 1993; Feligini et al., 1998; Rachagani Zimmermann et al., 1985; Ikeguchi et al., 1997). et al., 2006; Caroli et al., 2009) and mass spec- trometry (Criscione et al., 2009). Heterologous expression and purification of b-Lg from several species has also been described The first correct amino acid sequence of both from bacteria (Ariyaratne et al., 2002; bovine b-Lg was reported by Braunitzer et al. Ponniah et al., 2010) and yeast (Kim et al., 1997; (1972), and the position of one disulphide, Denton et al., 1998; Invernizzi et al., 2004). The 66–160, was identified unambiguously (Préaux methods used are generally ion exchange followed

218 L. Sawyer Fig. 7.2 Amino acid sequence variation within ruminant referred to explicitly: I in yak refers to Leu87 in cow becom- b-lactoglobulins relative to bovine genetic variant B. The ing Ile in the yak. The two disulphide bridges between resi- sequence is represented in single-letter notation with resi- dues in larger font indicating positions of genetic variation. dues 66–160 and 106–119 are shown with black hatching. Those variations referring to the domestic cow only have the variant letter: N in Dr refers to the Droughtmaster breed Yellow indicates regions of 310 helix, blue shows b-strands in which residue Asp28 is changed to Asn. Other species are and brown depicts a-helix. The figure is adapted from the one kindly provided by the Fonterra Research Centre, Palmerston North, New Zealand, with permission and Lontie, 1972). The other disulphide, 106– be of genetic origin. Interestingly, although poly- 119, with a free cysteine at position 121, remained morphisms have been detected in the coding uncertain until the crystal structure emerged regions of the goat gene, none leads to an amino (McKenzie and Shaw, 1972; Papiz et al., 1986; acid change (Ballester et al., 2005). Sawyer, 2003), the uncertainty eventually explained as urea-mediated disulphide inter- Many nonruminant b-Lgs have now been change (Phelan and Malthouse, 1994). sequenced, and their sequences diverge considerably from that of bovine b-Lg B and from one another. Figure 7.2 presents the differences relative to Many of the substitutions observed cannot have b-Lg B of the ruminant species that are synthe- arisen from single point mutations. As noted already, sised under the control of codominant alleles. the milk of dolphin, dog, cat, donkey, horse and pig b-Lg from a subset of Droughtmaster cattle, gly- contains b-Lg from more than a single gene, and cosylated at Asn28 (Bell et al., 1981b) as it is in indeed ruminant pseudogenes have been identified the related glycodelin, and minor truncated com- that appear to reflect these other genes (see above). ponents found only in the milk of Romagnola The complete sequences of the available mono- cattle (Zappacosta et al., 1998) are the only atypi- meric b-Lgs, together with a representative rumi- cal phenotypes, although the truncation may not nant sequence, are shown in Table 7.3. The data are

7 b-Lactoglobulin 219 Table 7.3 Amino acid sequences of b-lactoglobulin for the nonruminant species relative to that of bovine b-lactoglob- ulin B (continued)

220 L. Sawyer Table 7.3 (continued) The sequences of b-Lg from nonruminant species; the bovine B variant is included as representative of the ruminant proteins. Bovine and caprine pseudogenes, the human and macaque glycodelins (Gcn) are also included. The sequences are divided every ten residues. The bold regions of the cow sequence represent the lipocalin motifs defined by PRINTS (Attwood et al., 2003). The secondary structure observed for the lattice X form of bovine b-Lg is shown above the cow sequence: 3 = 310-helix; A,B,C, etc. = b-strand A,B,C, etc.; a = a-helix. Conserved residues are indicated if strictly con- served (*), similar (:) or broadly similar (.). The Swiss-Prot databank entries are cow—P02754; cow pseudogene (Passey and Mackinlay, 1995); goat pseudogene—Z47079; the author is indebted to Dr J. M. Folch for help with the translation; dolphin—B61590 (Pervaiz and Brew, 1987); pig—P04119; dog—P33685; dog III—P33686; cat I—P33687; cat III— P33688; donkey I—P13613; horse I—P02758; donkey II—P19647; horse II—P07380; cat II—P21664; baboon— AF021261; macaque (Hall et al., 2001); glycodelin—P09466; macaque glycodelin—Q5BM07; wallaby—Q29614; kangaroo—P11944; possum—Q29146; platypus–F65´48 mostly retrieved from the Swiss-Prot databases structurally conserved regions indicative of a core (The Uniprot Consortium, 2008). member of the lipocalin family (Flower, 1996). Although the glycosylated b-LgDr (Bell et al., A number of partial and complete cDNA 1981b) is atypical of ruminant species, Batt and sequences (Willis et al., 1982; Mercier et al., co-workers have produced a glycosylated bovine 1985; Gaye et al., 1986; Jamieson et al., 1987; b-Lg by mimicking the glycosylation sites of the Ivanov et al., 1988) were followed by complete related lipocalin, glycodelin (Kalidas et al., 2001), gene sequences (Ali and Clark, 1988; Alexander including the Asn28 of b-LgDr. The nonruminant et al., 1989) that revealed the pattern of introns sequences show much lower sequence homology subsequently found to be consistent among the (typically 30–70% identity), in keeping with their wider lipocalin family (Salier, 2000; Sanchez being the product of separate genes (Sanchez et al., 2006). et al., 2006). In passing, it should be noted that b-Lg contains all 20 amino acids in relative Examination of the primary structures of b-Lg amounts that make it valuable nutritionally. reveals no obviously repetitive or unusual stretches of sequence. There are, however, the

7 b-Lactoglobulin 221 7.6 Structure structure of the triclinic X form of the cow pro- tein (Brownlow et al., 1997) corrected the thread- Both macromolecular (X-ray) crystallography ing errors in the medium resolution structure and NMR spectroscopy have been widely applied (Papiz et al., 1986) and provided an independent to b-Lg, and it is convenient to describe the view of the dimer. Structures of crystal forms molecular structure of b-Lg here as a basis for with a monomer in the asymmetric unit require understanding the protein’s properties. The need the molecular twofold rotation axis to be coinci- for suitable crystals for the X-ray technique is dent with a crystallographic one (Bewley et al., obviously a limitation, although as far as can be 1997; Qin et al., 1998a, b). Detailed X-ray crys- judged, the structures obtained are generally a tallographic studies on the native/recombinant fair reflection of the solution state of the protein. protein inter alia have now been carried out on On the other hand, heteronuclear NMR spectros- several crystal forms of bovine b-Lg (Table 7.4), copy that produces a structure in solution, gener- as well as the pig and reindeer proteins, and it is ally requires a suitable over-expression system. with reference to these structures that we discuss Both techniques therefore have played an impor- the molecular properties of the protein. tant role in our understanding of the structure and properties of b-Lg. In solution, the experimental values for a-helix, b-sheet and random coil content around Although b-Lg was one of the first proteins to 8%, 45% and 47%, respectively, are broadly sim- be subjected to X-ray analysis (see Hodgkin and ilar to the values predicted from the sequence Riley, 1968), and low-resolution work on salted (see Sawyer, 2003). These values agree with what out forms, lattices X, Y and Z, was summarised by is observed in the crystal and NMR structures. Green et al. (1979), the first high-resolution However, most prediction methods indicate a significantly greater helical content than is Table 7.4 Crystal structure data for native b-lactoglobulins Resolution (Å) Space group Lattice codea Zb pH b-Lgc PDB coded References 2.8 B2212 Y 1 7.6 A Papiz et al. (1986) 1.7 P1 X 2 6.5 AB 1beb Brownlow et al. (1997) 1.8–2.0 C2221 Ye 1 7.6 A, B, C Bewley et al. (1997) 2.56 P3221 Z Qin et al. (1998a) 2.24 P3221 Z 1 6.2 A 3blg Qin et al. (1998a) 2.49 P3221 Z Qin et al. (1998a) 2.2 P3221 Z 1 7.1 A 1bsy Qin et al. (1999) 2.4 P3221 Z Hoedemaeker et al. (2002) 2.0 C2221 Y 1 8.2 A 2blg Oliveira et al. (2001) 1.95 C2221 Y Oliveira et al. (2001) 3.0 P212121 U¢ 1 7.1 B 1bsq Adams et al. (2006) 2.1 P1 Oksanen et al. (2006) 1 3.2 Pig 1exs 1 7.9 A 1qg5 1 7.9 B 1b8e 4 5.2 A 2akq 8 6.5 Reindeer 1yup 2.1 C2221 Y 1 7.4 A 2q2m Vijayalakshmi et al. (2008) 2.2 P3221 Z 1 7.5 B 3npo Loch et al. (2011) 2.0 P65 2 7.0 Gyubaf 3kza Ohtomo et al. (2011) aThe lattice code is that assigned by Aschaffenburg et al. (1965) bZ is the number of b-Lg monomers per asymmetric unit. Z = 1 means that the dimer has a strict crystallographic twofold rotation axis cThe b-Lg used cow unless otherwise stated. A, B, C refer to the cow b-Lg genetic variant dThe PDB code is that given to the atomic coordinates by the Protein Data Bank (http://www2.rcsb.org/pdb/home/home.do) eThis is the conventional setting of B2212, originally designated as lattice Y fGyuba is a chaemera made from strands of bovine b-Lg and the loops of the equine protein

222 L. Sawyer observed (Sawyer and Holt, 1993; Sakurai et al., swap of the N-terminal 12 residues so that, for 2009) which has significant implications for the example, Glu9 of subunit A binds to Thr142 of folding of the protein. The structure of the mono- subunit B. The bulk of the polypeptide chain, mer consists of nine strands of antiparallel however, follows the typical fold with b-strands b-sheet, eight of which wrap round to create a A–I in a similar relationship to those of the bovine flattened, conical barrel or calyx, closed at one protein. The major helix, too, is similarly arranged. end by Trp19. Strand A bends through about 90° The dimer formed by the chaemeric ‘Gyuba’ pro- around residues 21–22 to allow it to form an anti- tein closely resembles that of the cow and reindeer parallel interaction with strand H, thereby com- proteins since the core b-sheet has been retained, pleting the calyx. The calyx is approximately while the loop regions have been grafted from the cylindrical with a volume of 315 Å3 and a length equine protein (Ohtomo et al., 2011; ‘Gyuba’ is of some 15 Å and walls that are hydrophobic. It from the Japanese words for cow and horse). has been suggested that the calyx is empty unless occupied by a ligand (Qvist et al., 2008). A ninth Several analyses show the differences between strand, I, is on the outside, on the opposite side the A, B (and C) genetic variants (Bewley et al., of strand A to strand H, and so is able to form part 1997; Qin et al., 1999; Oliveira et al., 2001). The of the dimer interface which buries 570 Å2 on A/B sequence changes are Asp64Gly and each monomer. The interface involves antiparal- Val118Ala. The effects on the solution behaviour lel interactions of residues 146–150 with those of of these small changes are, however, significant the other subunit, together with Asp33, Ala34 and (Townend et al., 1964; Jakob and Puhan, 1992; Arg40 in the large AB loop, the Asp-Arg forming Hill et al., 1996; Manderson et al., 1998, 1999a, an essential inter-subunit ion pair (Sakurai and b) and appear to arise largely from the Val/Ala Goto, 2002). There is a 3-turn a-helix on the change at 118 in strand H creating a cavity, and outer surface of the calyx over strand H that is not hence less favourable packing, in the core. The in contact with its equivalent in the other subunit. destabilisation of the B variant relative to the A The polypeptide chain between the b-strands has been estimated to be around 5 kJ/mol includes two separate 310-turns and a g-turn, con- (Alexander and Pace, 1971; Qin et al., 1999), served in all lipocalins that have the –T97DY99– which is expected for the loss of two methyl sequence. The fold of the monomer is shown in groups (Shortle et al., 1990). Interestingly, a Fig. 7.3, which also shows the bovine dimer. hydrogen–deuterium exchange study suggests that the A variant is more flexible than the B vari- While the reindeer structure is essentially the ant (Dong et al., 1996). The Asp/Gly change same as that of the cow, the porcine structure occurs in the flexible external loop CD, and so its (Fig. 7.4) differs in a number of respects effect is less marked. In the C variant, the change (Hoedemaeker et al., 2002). Most obviously, the Gln59His is at the end of the C strand causing a dimerisation that occurs at low pH (cf. the cow redistribution of side-chain interactions that affect protein) is quite distinct and involves a domain the solution behaviour (Bewley et al., 1997). Fig. 7.3 (continued) rear of the picture is in the open posi- twofold axis is perpendicular to the plane of the page, the left-hand part of the molecule has been rotated about 90° tion in this structure. The drawing was made using PyMOL about a vertical axis, the right hand has been rotated simi- larly in the opposite direction. The stick representation of (2008). (b) The dimer of b-Lg showing the interaction the main chain surrounded by a semitransparent surface is shown with the area surrounding the AB loop and the I sites in magenta: the antiparallel arrangement of strands I strand indicating the contact surface which is also shown on the right-hand part of the figure. Notice that strand I together with residues in the AB loop, in particular Asp33 and the AB loop, in particular the Asp33–Arg40 interac- and Arg40. Also shown is the buried carboxyl from Glu89 tion, are the only points of contact between the monomers. on the EF loop in orange red, which is in the closed posi- The drawing was made using PyMOL (2008) tion. Notice that the EF loop in the right-hand, blue sub- unit has a break where the electron density was poor. The drawing was made using PyMOL (2008). (c) The dimer interface of b-Lg is shown ‘opened out’: If the molecular

7 b-Lactoglobulin 223 Fig. 7.3 (a) The monomer of b-lactoglobulin A viewed Ala, respectively, in the B variant. The disulphides 66–160 approximately down the molecular twofold axis. The Asp64 and Val118 residues are those changed to Gly and and 106–119 are shown together with the free Cys121. Tryptophans 19 and 61 are also shown. The EF loop at the

224 L. Sawyer Fig. 7.4 The dimer of porcine b-Lg showing the quite N-terminal regions are ‘domain swapped’, the blue resi- distinct dimer formation from the ruminant protein. The I dues interacting with the green subunit and vice versa. strands have no contacts shorter than 4 Å, and the two The drawing was made using PyMOL (2008) Crystallographic analyses can provide some 2, where the monomer predominates, simplify- information about the mobility or flexibility of ing the analysis. Similar results have also been the protein main and side chains from the B (or obtained by other groups (Belloque and Smith, temperature) factors: the larger the value, the 1998; Forge et al., 2000; Uhrinova et al., 2000; less well defined, or the more mobile, the atom. Edwards et al., 2002), the latter two using 13C- With b-Lg, several sections of the polypeptide and 15N-labelled recombinant b-Lg to refine the chain regularly appear to have high B factors, complete structure in solution at pH 2. The core and the corresponding electron density is weak of the protein is essentially the same as that and indistinct. Comparison of the various crystal described by the crystallographic analyses forms of the protein shows that some regions (Molinari et al., 1996; Ragona et al., 1997; (1–5, 32–38, 60–67, 112–116, 157–162) are less Fogolari et al., 1998; Belloque and Smith, well, or only poorly, defined; that is, they are 1998; Kuwata et al., 1998; Uhrinova et al., mobile. The EF loop, around 85–90, repositions 2000). However, the external loops and the itself in response to changes in pH, and compar- position of the helix are modified relative to the ing the left-hand panel of Fig. 7.5a (pH 6) with crystal structures at neutral pH (Jameson et al., the right-hand one (pH 8) shows this most con- 2002). Some ingenious protein engineering that vincingly. Figure 7.5b also shows the movement introduced an additional disulphide bridge of the EF loop in a comparison of the high-pH through Ala34Cys allowed NMR work to pro- crystallographic structure with the low-pH NMR ceed at neutral pH, once again confirming the structure. fold of the polypeptide chain (Sakurai and Goto, 2006). Protein flexibility is better observed in solu- tion by NMR methods. Using 1H NMR, Molinari No such monomer–dimer complication has and co-workers have painstakingly derived a dogged the NMR studies of the monomeric equine structure for the protein that in many features and porcine b-Lgs. Resonances for the native agrees with the crystal structures (Molinari equine structure have been determined (Kobayashi et al., 1996; Ragona et al., 1997; Fogolari et al., et al., 2000, 2002), but a final structure has not 1998). The NMR studies were carried out at pH been published although comparisons with bovine

7 b-Lactoglobulin 225 Fig. 7.5 (a) The structure of bovine b-lactoglobulin right-hand image is the same view but of the structure at pH 8 where the EF loop has swung away providing access viewed into the central ligand-binding calyx at the bottom to the binding site. The drawing was made using PyMOL (2008). (b) Showing a superposition of 40 NMR struc- of which is Trp19. In the left image of the structure at pH 6, tures at pH 2.5 with the high-pH (red) and low-pH (yel- the EF loop is in the closed position with the Glu89 buried low) X-ray structures of bovine b-lactoglobulin. Most and the side chain of Leu effectively occluding access to loops are clearly more mobile than the core sheet struc- ture, and the pH-dependent movement of the EF loop is the calyx. Notice that the side chains within the calyx are clearly visible (figure adapted from Uhrinova et al., 2000, with permission. Copyright 2000, American Chemical hydrophobic with the exception of Gln120. Met107 lies Society) closer to the viewer than Phe105, seen edge on, and these two side chains move to accommodate the ligand. The charged residues Lys60, Glu62 and Lys69 are at the entrance and can interact with polar head groups of ligands. The b-Lg show a high degree of consistency. Studies assignment is possible (Invernizzi et al., 2004). on the porcine protein have also shown that it is What is revealed so far is that the NMR structure monomeric at neutral pH (Ugolini et al., 2001; is consistent with that determined by X-ray crys- Ragona et al., 2003). Heterologous expression tallography. However, neither horse nor pig b-Lg has also been reported such that a full NMR NMR coordinate sets have yet been deposited.

226 L. Sawyer 7.7 Amino Acid Environments and Lötzbeyer, 2002). Another cross-linking approach has been to use a Ca2+-independent Probing the environments of the various amino microbial transglutaminase that does form poly- acid types was performed originally by protein mers of b-Lg (Hemung et al., 2009) but requires chemistry methods which, though specific for either heating or disulphide reduction of b-Lg amino acid type, could cause significant protein (Sharma et al., 2001; Eissa et al., 2006). The perturbation and therefore required careful inter- transamination of b-Lg with low molecular pretation (reviewed by Sawyer, 2003). The weight amines identifies the residues in an amine- advent of site-directed mutagenesis has added dependent manner. Thus, Gln 35, 59, 68 and 155 considerably to studies of the stability, reactivity are transaminated with 6-aminohexanoic acid and environment of individual amino acid resi- (Nieuwenhuizen et al., 2004) and Gln 13, 68, dues. Early work on the environment of specific 15/20 and 155/159 with 5-biotinamido-pen- amino acid types within bovine b-Lg was sum- tylamine (Hemung et al., 2009). marised by Townend et al. (1969) and found to be essentially correct when the X-ray structure The proximity of the potential quenchers cys- emerged (Brownlow et al., 1997; Bewley et al., tine 66–160 to Trp61 and Arg124 to Trp19 means 1997; Qin et al., 1998b). Although some studies that the local environments are sensitive to small have been carried out on the caprine and ovine changes in structure and to their accessibility by proteins, which from the sequence identities are quenchers (Busti et al., 1998; Bao et al., 2007; expected to be very similar to bovine, less was Harvey et al., 2007; Edwards et al., 2009). This known about the monomeric b-Lgs, until the in turn means that observed fluorescence changes, detailed NMR studies of the horse protein were generated, for example, by ligand binding, must performed (Fujiwara et al., 1999; Kobayashi be interpreted with caution. Lysine methylation, et al., 2000) and the crystal structure and NMR acetylation or succinylation has produced mate- details of the porcine protein published rial, far from native, that has antiviral activity (Hoedemaeker et al., 2002; Invernizzi et al., (Chakraborty et al., 2009; Sitohy et al., 2010), 2004; D’Alfonso et al., 2005). Some recent and Caillard et al. (2011) have discussed the use results on the reactivity of various amino acids of succinylated b-Lg as a suitable vehicle for are summarised below. oral drug delivery. Antiviral activity has also been shown for esterified b-Lg (Sitohy et al., The free cysteine, Cys121, in bovine b-Lg is an 2007) although at a level significantly lower than obvious target, and its pH-dependent availability acyclovir. and effect on dimer stability examined (Sakai et al., 2000; Chamani, 2006). Cys121 is some way Specific side-chain reactivity can also be from the interface, but its reaction will interfere explored by proteolysis, and the reader is referred with the helix transmitting an effect that usually to Volume 1B and Hernandez-Ledesma et al. destabilises the dimer. Disulphide interchange (2008) for details of the bioactive peptides that occurs under denaturing conditions leading to have been produced from b-Lg by a variety of aggregation (Creamer et al., 2004), an effect that enzymes and procedures. is affected by the genetic variant (Manderson et al., 1998, 1999a, b). Mutation of Cys121 desta- 7.8 Solution Studies bilises the structure somewhat but eliminates the disulphide interchange (Cho et al., 1994b; Yagi Essentially every available technique has been et al., 2003; Jayat et al., 2004). An alternative applied to probe the physicochemical behaviour cross-linking procedure using tyrosinase, and the of b-Lg in vitro. Almost all of these studies have modulator, caffeic acid, produces polymers with used the bovine protein and, unless specifically a molecular weight >300 kDa with the maximum mentioned, it is this protein which is being rate occurring between pH 4 and 5 (Thalmann described. The principal physicochemical param- eters are given in Table 7.5.

7 b-Lactoglobulin 227 Table 7.5 Selected molecular properties of bovine b-lactoglobulin Number of amino acids 162 http://www.expasy.ch/cgi-bin/protparam 2,596 http://www.expasy.ch/cgi-bin/proparam Total number of atoms (B variant) C817H1316N206O248S9 http://www.expasy.ch/cgi-bin/protparam 18,281.2 http://www.expasy.ch/cgi-bin/protparam Molecular formula (B variant) 18,278.8 Leonil et al. (1995) 5.407 Godovac-Zimmermann et al. (1996) Monomeric Mr (B genetic variant) (Da) 4.968 Godovac-Zimmermann et al. (1996) Mr (B genetic variant) (Da) 4.83 http://www.expasy.ch/cgi-bin/protparam Isoelectric point (B genetic variant, native) (pH) 0.961 Townend et al. (1960b) 0.919 http://www.expasy.ch/cgi-bin/protparam (Reduced and denaturing conditions) 0.46 Pessen et al. (1985) 2.04 Aymard et al. (1996) Theoretical isoelectric point (B variant) 3.19 Aymard et al. (1996) 2.1 Panick et al. (1999) Extinction coefficient: 1 mg/mL at 278 nm 3.44 Timasheff and Townend (1964) 2.83 Cecil and Ogston (1949) Calculated extinction coefficient 10.5 Aymard et al. (1996) 6.7 Aymard et al. (1996) Hydration (g H2O/g protein) 3.4 Tanford (1961) Monomer hydrodynamic radius (nm) 0.751 Svedberg and Pedersen (1940) 2:1 Green and Aschaffenburg (1959) Dimer hydrodynamic radius (nm) 3.07 × 10−3 M Sakurai et al. (2001) Radius of gyration, dimer (nm) 4.93 × 10−6 M Sakurai et al. (2001) 1.96 × 10−5 M Zimmerman et al. (1970) Radius of gyration, octamer (nm) 4.55 × 10−12 M3 Gottschalk et al. (2003) 698 (corrected for Mr) Ferry and Oncley (1941) Sedimentation coefficient (S°20w × 1013/s) Monomer diffusion coefficient (10−11 m2/s) Dimer diffusion coefficient (10−11 m2/s) Intrinsic viscosity (mL/g) Partial specific volume (cm3/g) Axial ratio (dimer) Dimer K (A genetic variant) d pH 3.0, 10 mM NaCl, 293 K pH 6.5, 20 mM NaCl, 293 K pH 8.2, 130 mM NaCl, 293 K Octamer dissociation constant pH 4.7, 274 K Dipole moment (Debye) 7.8.1 Solubility because of the unique distribution of surface charge, and hence dipole, at neutral pH (Ferry b-Lg, a globulin, is largely insoluble in distilled and Oncley, 1941), a view shared by Piazza water and so can be precipitated and even crys- et al. (2002) and Bertonati et al. (2007). This tallised by dialysis (Senti and Warner, 1948; conclusion has been given further weight in a Green et al., 1956; Adams et al., 2006). Salt systematic study of the solution properties (Holt increases the solubility quite dramatically: Polis et al., 1999). Solubility curves for b-Lg around et al. (1950) dissolved 1.8 g/L in water at the pI the pI (Grönwall, 1942) are often shown in compared to 16.5 g/L at pH 5.2 in 0.2 M NaCl, undergraduate biochemistry texts (e.g. Voet and a tenfold increase, and Treece et al. (1964) Voet, 2004). Salting out concentrated protein showed b-Lg B to be about five times more sol- solutions, the other extreme, is the standard way uble than b-Lg A, a result that is perhaps coun- of growing X-ray quality crystals of any b-Lg terintuitive on account of the A variant having (Aschaffenburg et al., 1965; Rocha et al., 1996; an extra charge (Asp for Gly64). Arakawa and Hoedemaeker et al., 2002; Oksanen et al., 2006), Timasheff (1987) maintain that because the sol- although dialysis against distilled water is an ubility is anomalous, much salt binding occurs alternative (Adams et al., 2006).

228 L. Sawyer reason may be some form of carboxyl–carboxy- 7.8.2 Molecular Size late interaction (Sawyer and James, 1982). Indeed, Armstrong and McKenzie (1967) showed Early studies of the Mr of bovine b-Lg under that carbodiimide modification of the carboxyls various conditions converged to a value of affects only the ability to octamerise. No crystals ~36,000 Da although at high dilution or low pH, capable of full X-ray structure determination of a half-size component became significant. The the A variant at pH 4.6 have been obtained so that association/dissociation data for bovine b-Lg are a direct view of the octamer remains elusive. summarised in Table 7.5, and fuller discussions Tetragonal crystal forms have been reported, can be found elsewhere (Verheul et al., 1999; from which it is tempting to speculate that they Sakurai et al., 2001; Gottschalk et al., 2003; may reflect the likely 422 symmetry (Green and Invernizzi et al., 2006; Bello et al., 2008, 2011; Aschaffenburg, 1959; Timasheff and Townend, Mercadente et al., 2012). Ruminant b-Lg at 1964; Witz et al., 1964). However, this need not neutral pH is mostly a dimer of two identical or be the case. Indeed, Mercadente et al. (2012) near-identical subunits and at pH 2 at low ionic have shown very recently that in 0.1M NaCl and strength the monomer species predominates, with 45 microM protein, the dimer is the predominant the core remaining pretty much in the native form over the pH range 2.5 to 7.5 at 25ºC with form, although both NMR and fluorescence no evidence of larger complexes. Interestingly, studies show subtle changes (Mills and Creamer, although neither determination was at pH 4.6, 1975; Molinari et al., 1996; Kuwata et al., 1999; the salt-free bovine (Adams et al., 2006) and the Uhrinova et al., 2000). Low salt concentration at reindeer (Oksanen et al., 2006) structures have low b-Lg concentrations enhances dissociation four and eight subunits in the asymmetric unit, (Aymard et al., 1996; Renard et al., 1998). For hinting at a larger structure than the dimer. [b-Lg] < 0.3 mM, the Kd in 0.2 M NaCl of Over the pH range 8–9.5, slow time-dependent 1.00 × 10−5 M rises to 3.55 × 10−3 M in the absence changes occur in b-Lg. At pH values above 8.5, of NaCl. Dissociation is also enhanced by thiol reversible dissociation occurs (Georges and modification (Burova et al., 1998) and increasing Guinand, 1960; Invernizzi et al., 2006) and, temperature (Aymard et al., 1996; Bello et al., above pH 9, the optical rotatory dispersion 2008, 2011). These recent Kd values are in keeping (ORD), circular dichroism (CD) and solubility with those derived elsewhere, which also show change with time as the protein denatures irre- that the b-Lg genetic variants dissociate in the versibly and aggregates (Groves et al., 1951; order A ³ B > C (Timasheff and Townend, 1961; Christensen, 1952; Herskovits et al., 1964; Thresher and Hill, 1997; Bello et al., 2011). Townend et al., 1967). Addition of a thiol-block- It has also been shown by Hill et al. (1996) that ing group can inhibit aggregation, implicating AA, BB and AB have K values of 1.5, 1.8 and thiol oxidation and/or thiol/disulphide exchange d 2.1 × 10−6 M in simulated milk ultrafiltrate buffer, in the formation of the heavier components (Roels pH 6.6 and 20°C. Recent work on the energetics et al., 1966). of hydration involving both experimental and As regards the nonruminant protein, compara- theoretical studies of dimer formation (Bello tive pH studies by mass spectrometry on the pig et al., 2008) has shown that subunit association and cow b-Lgs (Invernizzi et al., 2006) find that also involves ‘burying’ some 36 water molecules. the pig protein associates to a dimer distinct from Between pH 3.5 and 6.5, with a maximum at that of the ruminants (Hoedemaeker et al., 2002) pH 4.5, the bovine A variant forms octamers (i.e. at pH 4.0 but is monomeric at pH 6.0 and above. four dimers), especially at low temperatures The K is 9 mM which is consistent with the d (Townend and Timasheff, 1960; Timasheff and 56 mM obtained by gel permeation at pH 3.0 Townend, 1961; Pessen et al., 1985; Verheul (Ugolini et al., 2001). Information on the equine et al., 1999; Gottschalk et al., 2003). Since the A protein indicates that it does not form dimers variant has the external Gly64Asp mutation, the between pH 3.3 and 8.7 although incubation at

7 b-Lactoglobulin 229 pH 1.5 for 2 h or treatment with 3 M urea at pH in the same region of the protein, albeit on the 8.7 was shown to produce some material with an surface. No change is detected in the IR spectrum apparent MW greater than the monomer (Ikeguchi (Casal et al., 1988). The changes have also been et al., 1997; Fujiwara et al., 2001). A chaemeric identified by NMR (Fogolari et al., 1998, 2000; form of the protein, retaining the cow core but Kuwata et al., 1999) and are generally fairly with the horse loops, has been shown to form small. The core remains compact although the cow-like dimers (Ohtomo et al., 2011). helix moves slightly relative to its position at neutral pH. Loops AB and CD adopt different 7.9 Conformation and Folding positions, and loop EF is in the ‘closed’ position, preventing access to the central calyx. Glu89 is Early work using CD and the related technique of buried. Presumed movement of the helix caused ORD together with ultracentrifugation and other by modification of the free Cys121 also leads to techniques (reviewed by Sawyer, 2003) showed dissociation of the dimer (Zimmerman et al., that bovine b-Lg underwent three pH-dependent 1970; Iametti et al., 1998; Burova et al., 1998), conformational transitions between pH 2 and 10 but whether dissociation at low pH causes the which can be summarised as: movement or results from it is not yet clear. Q↔N↔R→S 7.9.2 N ↔ R This pH-dependent conformational variation Between pH 6.5 and 7.8, the second reversible has been extended using ultrasonic, densim- conformational change (N ↔ R), often called etric and spectroscopic studies (Taulier and the Tanford transition, is observed (Groves Chalikian, 2001) which suggest that there are et al., 1951; Tanford et al., 1959). In bovine b- five distinct changes in conformation that lead Lg, this transition can be detected by a simple to variations in protein hydration, compressibility change in optical rotation ([a]D is −25° at pH 6, and specific volume, the extra transitions over but −48° at pH 8), by a decrease in the sedimen- the above scheme being below pH 2 and above tation coefficient (3.2–2.6 S) or possibly even a pH 10. thermal denaturation peak (de Wit and Klarenbeek, 1981; Qi et al., 1995, 1997). The 7.9.1 Q ↔ N change in sedimentation coefficient may result from protein expansion, shape variation (Tanford Bovine b-Lg variants A, B and C undergo the et al., 1959; Timasheff et al., 1966b) or increased reversible Q ↔ N transition between pH 4 and 6 dissociation (Georges et al., 1962; McKenzie (Timasheff et al., 1966b; McKenzie and Sawyer, and Sawyer, 1967). 1967). The pH-dependent increase in sedimenta- tion coefficient correlates with a contraction of Upon increasing the pH, the buried carboxyl the protein. The titration behaviour is consistent of the conserved Glu89 becomes exposed and with a two-proton ionisation for b-Lg A while b- ionised, with a positive enthalpy (Tanford and Lg B and b-Lg C follow a single proton transi- Taggart, 1961), arising from hydrogen bonding to tion. This is in keeping with the extra Asp64 in the the carbonyl of Ser116 (Brownlow et al., 1997; A variant. In b-Lg C, one extra cationic residue Qin et al., 1998a). The anomalous carboxyl per subunit, presumably His59, is exposed upon (pKa = 7.3), which titrates normally in urea- and increasing the pH. No aromatic residues are alkali-denatured b-Lg (Tanford et al., 1959), has involved in this transition (Timasheff et al., also been observed in caprine b-Lg (Ghose et al., 1966a; Townend et al., 1969) which is slightly 1968) and appears to be a conserved feature of surprising since His59, Trp61 and Asp64 are located b-Lgs since the residue is conserved in all spe- cies, except the fur seal in which it is Gln (Table 7.3; Ragona et al., 2003; Cane et al., 2005;

230 L. Sawyer Edwards et al., 2009). A Tyr has been shown to Glu108 which in turn allows strand D and loops EF and GH to rearrange, thereby allowing access to be involved (Townend et al., 1969), and Tyr102 is the internal binding site. By varying the humidity in the vicinity of Glu89 although no significant of the crystals, Vijayalakshmi et al. (2008) have movement is observed between N and R states produced a form of b-Lg in which the EF loop in one subunit is closed but open in the other, from (Qin et al., 1998a). Cys121 also becomes more which it appears that the Tanford transition does accessible to pCMB (Dunnill and Green, 1965) not involve inter-subunit cooperativity. and KAu(CN)4 (Sawyer and Green, 1979) when Ragona et al. (2003) have shown that for por- the pH is increased from 6 to 8, but it is some way cine b-Lg, binding does indeed occur but only at pH values above 8.6. The pKa of Glu89 was mea- from Glu89 and the EF loop. That the change sured at 9.7, and the calculated value from the affects access to the calyx has been shown by the porcine structure is 7.4. In the light of this obser- vation, it would be interesting to measure the pH careful binding studies of Ragona et al. (2003) dependency of ligand binding in other b-Lgs monitoring the NMR signal from 13C-labelled reported not to bind ligands (Pérez et al., 1993). palmitic acid. The bound fraction is pH depen- 7.9.3 R → S dent with a half-site occupancy at about pH 5.5, The third, irreversible, conformational change is two whole pH units below the H+-mediated the alkali denaturation of b-Lg observed by many (Groves et al., 1951; Townend et al., 1960a; Tanford transition, but corresponding to the cal- Timasheff et al., 1966a; Hui Bon Hoa et al., 1973; Purcell and Susi, 1984; Casal et al., 1988; culated pKa of Glu89 in the ‘closed’ N state. The Mercadé-Prieto et al., 2008). pH titration behaviour compared to the ligand- 7.9.4 Folding binding behaviour indicates an interesting shift in The ‘folding problem’ in biology is one that has the pKa of Glu89 presumably brought about by the fascinated scientists for many years: given an sparingly soluble ligands binding to the small amino acid sequence, can one predict its native 3D structure? (Richards, 1991) Because b-Lg is a concentration of open form present at pH below relatively small b-barrel protein, it is a useful subject for such studies. Further, the existence of 7. Recall that the calyx is apparently empty (Qvist extra helical segments observed during the refold- ing makes the reason for the switch from helix to et al., 2008). No ligand binding is observed at pH sheet all the more intriguing. It is worth noting that essentially all folding studies are performed 2 (Ragona et al., 2000). Molecular dynamics by refolding the unfolded protein, usually by diluting the urea or guanidinium chloride used to simulation of the protonated and deprotonated achieve unfolding. Thus, it is important that suit- able conditions are found which permit refolding Glu89 is consistent with the above behaviour to the native state. With b-Lg such conditions are (Eberini et al., 2004) readily attainable although a great deal of work over the years has centred round the irreversible Recently, Goto and his co-workers have pro- denaturation that is important commercially. vided a detailed explanation of the Tanford transi- tion (Sakurai and Goto, 2006, 2007; Sakurai et al., 2009). Using the Ala34Cys mutant that stabilises the b-Lg dimer, three distinct phases were identified by NMR on the nano- to millisecond timescale. First, chemical shift differences revealed that a group associated with the G strand has a pKa of 6.9 which they assigned to Glu89. Next, on the microsecond timescale, consideration of the relax- ation data allowed significant fluctuations of the hydrogen bonds of Ile84, Asn90 and Glu108 to be identified above pH 7.0. These residues are at the hinge of the EF loop. On still longer a timescale, differences in signal intensity associated with resi- dues in the EF and GH loops together with some on the D strand correlate with the pH change. Together, these data were interpreted as initially Glu89 is deprotonated allowing fluctuation of the hydrogen bonding of residues Ile84, Asn90 and

7 b-Lactoglobulin 231 Reversible unfolding–refolding of bovine b- Fig. 7.6 A scheme showing the stages involved in the Lg at ambient temperatures has been examined at proposed folding/reversible unfolding (bold—a, b, c, d) low pH (Tanford and De, 1961; Pace and Tanford, and irreversible denaturation (e, f, g, h) of bovine b-lacto- 1968; Alexander and Pace, 1971; Hamada and globulin. It is probable that the dimer can undergo some Goto, 1997; Ragona et al., 1999) and at neutral unfolding/denaturation without first becoming a mono- pH (McKenzie et al., 1972; Hattori et al., 1993; mer, and certainly non-covalent interactions are present in Creamer, 1995; Subramaniam et al., 1996). The the various aggregates and fibrils. The molten globule methods used to monitor the process include state can be isolated by changing the dielectric constant, spectrophotometry, CD, NMR, fluorescence, but is most probably a transient state in the normal folding DSC, ligand-binding and antibody recognition. pathway The problem of disulphide interchange identified by McKenzie et al. (1972) was addressed further studies on caprine b-Lg have shown it to be by Cupo and Pace (1983) who used a thiol- slightly less stable than either bovine b-Lg A or modification approach to show that the extra, B (Alexander and Pace, 1971). external disulphide destabilised the structure. Sakai et al. (2000) showed that a modified Cys121 Work with equine b-Lg, a monomer under produced a molten globule-like structure at pH physiological conditions, has identified both a 7.5 while at pH 2.0 the protein remained native- reversible molten globule state and one in which like. Creamer (1995) minimised possible inter- there is also a greater-than-native content of change by working rapidly at pH 6.7, and his a-helix at pH 1.5 (Ikeguchi et al., 1997; Fujiwara study showed the stabilising effects of added et al., 1999; Kobayashi et al., 2000). Ikeguchi and ligand. To address the folding problem in more colleagues have been able to equate the molten structural detail, SAXS, fluorescence, NMR, CD globule state with the burst phase state found with and proton exchange studies have all been used bovine b-Lg, and they observed protection from (Kuwajima et al., 1987; Hattori et al., 1993; hydrogen exchange of residues on strands A, F, G Hamada et al., 1995, 1996; Hamada and Goto, and H, and on the helix. Their more recent studies 1997; Arai et al., 1998; Kuwata et al., 1998, also reveal the presence of non-native helix at 2001; Mendieta et al., 1999; Ragona et al., 1999; alkaline pH as well as at low temperatures Forge et al., 2000). A refolding scheme has (Nakagawa et al., 2006, 2007; Matsumura et al., emerged whereby b-strands F, G and H and the 2008). They too have relied both upon stabilisa- main a-helix are formed within 2 ms with con- tion of intermediates in water–alcohol solvents comitantly, some non-native a-helix around the (e.g. Matsumura et al., 2008) and site-directed N-terminal part of the A strand. Thus, the helix mutagenesis (Yamada et al., 2006; Nakagawa and sheet E–H form first together with the et al., 2006, 2007) to study the folding. C-terminal half of the A strand, before the N-terminal part of the A strand with strands B–D Porcine b-Lg has been examined by Molinari completes the folding. Sakurai et al. (2009) sug- and her co-workers who have also identified an gest that the non-native helix clearly identified in intermediate state with non-native helix predictions is a means of preventing unwanted (D’Alfonso et al., 2005; Ugolini et al., 2001). hydrogen bonding during folding. They sum- They interpret the unfolding, as with the bovine marise the folding of b-Lg in terms of an unfolded state passing through intermediate states with identifiable non-native helix, to produce, eventu- ally, the N state. It is not clear that the molten globule state is on the folding pathway, since most reports of its existence involve variation in temperature or solution dielectric. Figure 7.6 summarises this proposed scheme. Unfolding

232 L. Sawyer and equine proteins, as a process requiring inter- et al., 1997; Carrotta et al., 2003; Creamer et al., mediates, with the porcine protein being less sta- 2004; Bhattacharjee et al., 2005; Tolkach and ble at both pH 2 and pH 6 than bovine b-Lg. Kulozik, 2007), cold (Katou et al., 2001; Davidovic et al., 2009), pressure (e.g. Iametti 7.10 Denaturation et al., 1997; Belloque et al., 2000; Considine et al., 2007), organic compounds (e.g. D’Alfonso Arguably the largest topic discussed in the bovine et al., 2002; Dar et al., 2007), metal ions (e.g. b-Lg literature concerns its denaturation on its Stirpe et al., 2008; Gulzar et al., 2009) or metal own, but increasingly in the past couple of surfaces (Changani et al., 1997; Jun and Puri, decades, in mixtures with other proteins, carbo- 2005; Bansal and Chen, 2006). Studies with hydrates and lipids. This enormous literature combinations of these are also common (e.g. reflects the commercial importance of the effects Aouzelleg et al., 2004). Even various forms of of the various processing techniques on whole radiation can be used. For example, Bohr and milk, skim milk and whey protein preparations in Bohr (2000) have examined the effects of micro- which b-Lg is seen to play a crucial role. Indeed, wave radiation by SAXS suggesting that the as recently pointed out by de Wit (2009), a effects are nonthermal which is in keeping with significant amount of the recent literature merely the observation that microwave treatment at a repeats studies done before much of the elec- non-denaturing temperature enhances suscepti- tronic archive, which often begins only in mid- bility to proteolysis (Izquierdo et al., 2007). 1990s. However, cursory literature surveying is Similarly, the aggregation by g-radiation fol- not the only reason for revisiting the measure- lowed by SAXS is significant in solution, though ments, since monitoring techniques have become not in the solid state, and increases with decreas- significantly more sensitive which has high- ing protein concentration (Oliveira et al., 2006). lighted the fact that the behaviour of b-Lg is sen- It appears that the cross-linking initiated by OH• sitive to small changes in the conditions, radicals is through tyrosine side chains, most of especially at the protein concentrations that occur which are solvent accessible (Townend et al., in milk (Qi et al., 1995, 1997; Holt et al., 1998, 1969; Brownlow et al., 1997). 1999). In this section, ‘denaturation’ is taken to mean the generation, often irreversibly, of insolu- It is not yet clear in molecular detail how ble material. each of these denaturing agents acts to yield insoluble aggregates, although it is probable that Briefly, bovine b-Lg appears to denature the mechanism is agent-dependent and that sev- through an initial dissociation from dimer to eral of the stages may be common (Fox, 1995; monomer followed by a change in the polypep- Qi et al., 1997; Manderson et al., 1998, 1999a, tide chain conformation and subsequent aggre- b; Edwards et al., 2009; see also Volume 1B). gation. Because of the free thiol in b-Lg, The effects are also modulated by the presence disulphide interchange can also occur leading to of ligands (e.g. Boye et al., 2004; Considine oligo-/polymer formation, involving the 66–160 et al., 2005; Busti et al., 2005), generally thought cystine which is generally the more accessible, to stabilise the folded protein. What has become although intra-subunit exchange also can occur clear is that the mechanism depends on the pH, (McKenzie and Shaw, 1972; Manderson et al., the ionic strength and the nature of the ions, the 1999b; Creamer et al., 2004). Depending upon concentration and purity of the protein, the the precise conditions used, particulate or fibrous dielectric constant and the temperature (Dufour material can be formed and recently, amyloid and Haertlé, 1993; Li et al., 1994; Relkin, 1996; fibrils have been made to form most efficiently Renard et al., 1998; Foegeding, 2006; Krebs through low pH heating (Foegeding, 2006; et al., 2007). Distinguishable effects also derive Hamada et al., 2009). The denaturant can be from the genetic variant (Hill et al., 1996; alkali (Mercadé-Prieto et al., 2008), heat (e.g. Qi Manderson et al., 1998, 1999a, b; Holt et al., 1998). An attempt to illustrate the various stages

7 b-Lactoglobulin 233 that have been identified both in the unfolding– The number of individual ligands reported to refolding and the denaturation processes that bind to b-Lg is now probably well in excess of can lead to b-Lg coming out of solution is shown 200, many of the recent reports arising because in Fig. 7.6. The conformational changes dis- of the increased interest in the use of the protein cussed above are also implied since the denatur- as a means of trapping labile molecules (e.g. ation occurs more readily, the more open is the Loveday and Singh, 2008) and volatile flavours structure. and aromas in food (Kühn et al., 2006; Guichard, 2006). Table 7.5 of Sawyer (2003) contained The situation is somewhat different with the the majority of ligands identified before 2000 monomeric equine and porcine proteins that lack by methods such as equilibrium dialysis, a free thiol. Indeed, there is little published on the fluorescence measurements both intrinsic and denaturation of the nonruminant b-Lgs, although extrinsic, gel permeation/affinity chromatography, there is on their unfolding and refolding. The NMR and ESR. The past decade has seen those equine and porcine proteins are not stable in acid 111 entries for 76 distinct ligands increase rap- solution, unlike the bovine protein (Ikeguchi et al., idly, and so no such exhaustive table is included 1997; Ugolini et al., 2001; Burova et al., 2002; here; rather Table 7.6 provides a snapshot, giv- Invernizzi et al., 2006; Ohtomo et al., 2011). ing examples of the methods employed, the Yamada et al. (2005) have compared the heat- diversity of the ligands and the variation in bind- denatured state of equine b-Lg with that obtained ing constants from the supra-millimolar to the in acid, finding them to be similar but distinct sub-micromolar. Another significant table of from the cold-denatured state which, by CD, ligand-binding information can be found in SAXS and ultracentrifugation, appeared to be Tromelin and Guichard (2006). expanded and chain-like with more a-helix than the more compact acid-denatured protein, not Free fatty acids bind to bovine b-Lg in a man- unlike the intermediates of the bovine protein. ner dictated by the chain length, with the tightest binding being for palmitate (Spector and Fletcher, As one of the consequences of protein dena- 1970). Direct X-ray crystallographic evidence for turation is the formation of precipitate, in addi- ligand binding came relatively recently if one tion to food scientists, the process has recently neglects the heavy atom compounds used for attracted the attention of the nanotechnology phase determination (Green et al., 1979) and the (Krebs et al., 2009; Hirano et al., 2009) and soft homology model of Papiz et al. (1986), bromodo- matter physics (Donald, 2008) communities. decanoic acid (Qin et al., 1998b), palmitic acid (Wu et al., 1999) and retinol (Sawyer and 7.11 Binding Studies Kontopidis, 2000) which showed that they bind within the central calyx between the two b-sheets. The first report of a ligand bound to b-Lg appears Examination of the cavity shows that there is little to be that of oleic acid by Davis and Dubos room for a ligand longer than palmitate. Careful (1947), followed a few years later by Groves NMR studies with palmitate by Ragona et al. et al. (1951) who observed that 2 mol/mol (2000, 2003) and Konuma et al. (2007) confirmed (36 kDa) SDS were bound to the protein in a this internal binding site for palmitate. Loch et al. manner that stabilised it against thermal denatur- (2011) have recently provided both crystallo- ation. It was not until the 1960s, however, that graphic and solution data that show C8 and C10 binding constants began to be determined fatty acids bind to the calyx. Figure 7.7a shows (Wishnia and Pinder, 1966) by equilibrium dialy- palmitate (C16) bound in the calyx from which it sis, and then Futterman and Heller (1972) using can be seen that there is a degree of flexibility in fluorescence happened upon the retinol-binding two of the hydrophobic residues lining the pocket, ability, unaware of the family relationship Phe105 and Met107. This flexibility is mirrored in between retinol-binding protein (RBP) and b-Lg the binding of the carboxylate head groups shown which only emerged a decade later. in Fig. 7.7b which superimposes the binding of