Yilin Hu

Abstract

β-Casein is the major component (~80%) of the human casein system. Interacting with calcium and phosphates through its phosphorylated N-terminus, β-casein participates in the formation of micelles which carry these otherwise insoluble nutrients indispensable for the growth and development of newborns. Human β-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 β-casein, especially its N-terminal portion will provide a better understanding of the structure-function relationship of milk micelles.

To elucidate the role that the N-terminus of the protein plays in its folding, two recombinant human β-casein proteins have been expressed and purified from E.coli. One recombinant form, m+4, has four extra amino acids (GSHM-) at the N-terminus of human P-casein; and the other, m, has the exact amino acid sequence of the native protein.

Results from turbidity, sedimentation velocity and laser light scattering studies show that m+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 OP 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, m+4 shows some properties which are very different from the native OP. 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 nonpolar 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 OP and the two recombinants, m+4 and m, 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 m+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 characterstic 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.

LLU Discipline

Biochemistry

Department

Biochemistry

School

Graduate School

First Advisor

Charles W. Slattery

Second Advisor

Dan Borchardt

Third Advisor

E. Clifford Herrmann

Fourth Advisor

Satish M. Sood

Fifth Advisor

Lawrence C. Sowers

Degree Name

Doctor of Philosophy (PhD)

Degree Level

Ph.D.

Year Degree Awarded

1999

Date (Title Page)

12-1999

Language

English

Library of Congress/MESH Subject Headings

Caseins; Recombinant Proteins -- chemistry; Milk, Human.

Type

Dissertation

Page Count

xii; 191

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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Included in

Biochemistry Commons

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