| beta-Casein is the major component (∼80%) of the human casein system. Interacting with calcium and phosphates through its phosphorylated N-terminus, beta-casein participates in the formation of micelles which carry these otherwise insoluble nutrients indispensable for the growth and development of newborns. Human beta-casein or fragments thereof, are also found to have biological effects such as enhancement of calcium absorption. So far, knowledge of micelle formation is limited and structural studies of beta-casein especially its N-terminal portion will provide a better understanding of the structure-function relationship of milk micelles.;Results from turbidity, sedimentation velocity and laser light scattering studies show that rn + 4 has a monomeric molecular weight of around 22 kDa, which is very close to that of its native counterpart. In low salt buffer, it behaves similarly to the native 0P protein in that it undergoes a possible conformational change at a certain temperature, which leads to the subsequent significant aggregation of the molecule. On the other hand, rn + 4 shows some properties which are very different from the native 0P. Some minor self-association of the molecule starts at a lower temperature (10--15°C), the transition temperature where aggregation takes place is lower than that of the native protein and this protein seems to have a stronger tendency toward aggregation. During the conformational change at the transition point, the tryptophan residue shifts to a less non-polar environment and its mobility is more restricted as compared with the native protein, indicating the different environment in which the tryptophan residue resides.;Comparison of data obtained from turbidity studies of the native 0P and the two recombinants, rn + 4 and rn, suggests the possible interaction of the four extra amino acids with the calcium binding sites as well as the tentative role calcium plays in assisting the protein to fold into the "correct" conformation.;The rn + 4 protein seems to attain a conformational equilibrium by repeated heating and cooling, or a temperature cycling, through which it eventually stabilized in a pattern characteristic of the native protein. This equilibrium process seemed to be aided by the presence of Ca2+. The fact that it finally stabilized in a certain conformation again implies that the native protein normally folds into a particular conformation, aided for the recombinant mutant by temperature cycling.;To elucidate the role that the N-terminus of the protein plays in its folding, two recombinant human beta-casein proteins have been expressed and purified from E. coli. One recombinant form, rn + 4, has four extra amino acids (GSHM-) at the N-terminus of human beta-casein and the other, rn, has the exact amino acid sequence of the native protein. |
