Effect of Temperature on Self-Assembly of Bovine -Casein above and below Isoelectric pH. Structural Analysis by Cryogenic-Transmission Electron Microscopy and Small-Angle X-ray Scattering Christian Moitzi, †,‡ Irina Portnaya, § Otto Glatter, † Ory Ramon, § and Dganit Danino* ,§ Institute of Chemistry and Physical Chemistry, UniVersity of Graz, Graz A-8010, Austria, and Department of Biotechnology and Food Engineering and the Russell Berrie Nanotechnology Institute, Technion-Israel Institute of Technology, Technion City, Haifa 32000, Israel ReceiVed September 10, 2007. In Final Form: December 19, 2007 -Casein is one of the main proteins in milk, recently classified as an intrinsically unstructured protein. At neutral pH, it is composed of a highly polar N-terminus domain and a hydrophobic C-terminus tail. This amphiphilic block- copolymer-like structure leads to self-organization of the protein monomers into defined micelles. Recently, it has been shown that at room temperature, -casein also self-organizes into micelles in an acidic environment, but the effect of temperature on the micelles’ formation and properties at the low pH regime were not explored. In the present study, we used two complementary techniques, cryogenic-transmission electron microscopy (cryo-TEM) and small-angle X-ray scattering (SAXS), to characterize at high-resolution the micelles’ shape, dimensions, and aggregation numbers and to determine how these properties are affected by temperature between 1 and 40 °C. Two different regimes were studied: highly acidic pH where the protein is cationic, and neutral pH, where it is anionic. We found that flat disk-like micelles with low aggregation numbers formed at low temperature in the two pH regimes. Close to neutral pH increase in temperature involves a transition in the micelles’ shape and dimensions from flat disks to bulky, almost spheroidal micelles, coupled with a sharp increase in the micelles’ aggregation number. In contrast, no effects on the micelles’ morphology or aggregation number were detected in the acidic environment within the entire temperature range studied. The self-organization into disk micelles and the lack of effect of temperature in the acidic environment are linked to the unstructured character of the protein and to the charge distribution map. The latter indicates that below the isoelectric pH (pI), -casein loses the distinct separation of hydrophobic and hydrophilic domains, thereby suggesting that it may no longer be considered as a classical head-tail block-copolymer amphiphile as in neutral pH. Introduction -Casein is a ∼24 kDa calcium-sensitive phosphoprotein consisting of 209 amino acids, without disulfide cross-links. 1 It is highly amphiphilic and self-assembles into micelles at neutral pH values, 2-4 as well as below its isoelectric pH (pI). 5 The tendency of -casein to self-organize above the pI is related to a dominant, highly hydrophobic C-terminus 6 and a highly polar negatively charged N-terminal domain, whose first 21 amino acid residues contribute most of its net charge. 7 -Casein has been classified as a member of the natively denaturated/unfolded or intrinsically unstructured/disordered family of proteins (IUPs). 8 The intrinsically unstructured/ disordered regions are linked to a distinctive amino acid composition, characterized by a high content of proline, glutamine, and glutamic acid and the depletion of cysteine. 9,10 IUPs can exist either in a random coil conformation or in a conformation similar to a molten globule state, where some degree of secondary structure is preserved. 11 IUPs with a random coil conformation can further be divided into two major groups. One is an extended conformation typical of a random coil in a poor solvent, with little or no secondary structure. The other has a conformation similar to a premolten globule, where some residual secondary structure exists, and the protein state is between that of a random coil and that of a molten globule. 10-12 Recent studies of -casein at neutral pH suggest that it has a relatively high residual secondary structure, 9,13,14 and further, that upon micellization, it undergoes a transition from a permolten globule state (characteristic of the monomer) to a molten globule conformation. 15 It was previously assumed that only proteins with a folded conformation possess specific biological functions. Therefore, most past studies of caseins were performed above the pI, around neutral pH, where the stable casein milk micelles exert biological * Corresponding author. E-mail: dganitd@tx.technion.ac.il; tel.: +972- 4-829-2143; fax: +972-4-829-3399. † University of Graz. ‡ Current address: Department of Physics, University of Fribourg, Fribourg, Switzerland. § Technion-Israel Institute of Technology. (1) Ribadeau-Dumas, B.; Brignon, G.; Grosclaude, F.; Mercier, J. C. Eur. J. Biochem. 1972, 25, 505-514. (2) O’Connell, J. E.; Grinberg, V. Y.; de Kruif, C. G. J. Colloid Interface Sci. 2003, 258, 33-39. (3) de Kruif, C. G.; Grinberg, V. Y. Colloids Surf., A. 2002, 210, 183-190. (4) Mikheeva, L. M.; Grinberg, N. V.; Grinberg, V. Y.; Khokhlov, A. R.; de Kruif, C. G. Langmuir 2003, 19, 2913-2921. (5) Portnaya, I.; Ben-Shoshan, E.; Ramon, O.; Cogan, U.; Fass, D.; Danino, D. J. Agric. Food Chem., in press. (6) Berry, G. P.; Creamer, L. K. Biochemistry 1975, 14, 3542-3545. (7) Swaisgood, H. E. Chemistry of the caseins. In AdVanced Dairy Chemistry; Fox, P., McSweeney, P. L. H., Eds.; Kluwer Academic/Plenum: New York, 2003; pp 139-201. (8) Tompa, P. Trends Biochem. Sci. 2002, 27, 527-533. (9) Syme, C. D.; Blanch, E. W.; Holt, C.; Jakes, R.; Goedert, M.; Hecht, L.; Barron, L. D. Eur. J. Biochem. 2002, 269, 148-156. (10) Uversky, V. N. Eur. J. Biochem. 2002, 269,2-12. (11) Dunker, A. K.; Lawson, J. D.; Brown, C. J.; Williams, R. M.; Romero, P.; Oh, J. S.; Oldfield, C. J.; Campen, A. M.; Ratliff, C. R.; Hipps, K. W.; Ausio, J.; Nissen, M. S.; Reeves, R.; Kang, C. H.; Kissinger, C. R.; Bailey, R. W.; Griswold, M. D.; Chiu, M.; Garner, E. C.; Obradovic, Z. J. Mol. Graphics Model. 2001, 19, 26-59. (12) Receveur-Brechot, V.; Bourhis, J. M.; Uversky, V. N.; Canard, B.; Longhi, S. Proteins 2006, 62, 24-45. (13) Farrell, H. M.; Wickham, E. D.; Unruh, J. J.; Qi, P. X.; Hoagland, P. D. Food Hydrocolloids 2001, 15, 341-354. (14) Qi, P. X.; Wickham, E. D.; Farrell, H. M. Protein J. 2004, 23, 389-402. (15) Farrell, H. M., Jr.; Qi, P. X.; Uversky, V. N. New views of protein structure: Applications to the caseins: Protein structure and functionality. In AdVances in Biopolymers; Fishman, M. L., Qi, P. X., Wicker, L., Eds.; ACS Symposium Series 935; American Chemical Society: Washington, DC, 2006; pp 52-70. 3020 Langmuir 2008, 24, 3020-3029 10.1021/la702802a CCC: $40.75 © 2008 American Chemical Society Published on Web 03/08/2008