Masters Theses

Date of Award

12-1997

Degree Type

Thesis

Degree Name

Master of Science

Major

Biochemistry and Cellular and Molecular Biology

Major Professor

Elizabeth E. Howell

Committee Members

Daniel M. Roberts, Cynthia B. Peterson

Abstract

There is an idea in the literature that some multidomain proteins have evolved from the insertion of one protein into the primary sequence of another, parent protein resulting in a multifunctional protein. A multidomain protein, DHFR-CaM, was created by inserting calmodulin (CaM) into a surface &beta-bulge region of Escherichia coli dihydrofolate reductase (DHFR). Although the exact function of β-bulges is not known. it is thought that β-bulges have evolved to accommodate insertions and deletions and to help position residues of distant active sites. The latter idea was demonstrated by mutational studies of second site suppressor mutants of the D27S lesion in E. coli DHFR. The activity of the D27S mutant was found to be greatly reduced at pH 7.0, consistent with this residue being critical in catalysis. Partial suppressors of the D27S mutation have been localized to the area of the surface β-bulge of DHFR indicating that this area is important in protein function. Previous studies with the BB mutant, a mutant DHFR with a six amino acid insert in the surface (&bulge;-bulge, have shown that this region is also tolerant of insertions. With its demonstrated ability to accommodate insertions in this region and the great deal of information known about DHFR, this enzyme is ideal for the study of domain insertion mutants.

Equilibrium unfolding studies of the domain insertion protein, DHFR-CaM, were done to investigate the structural stability of this mutant. Three methods of monitoring the unfolding of this protein were utilized. Enzyme activity of the DHFR domain was used to monitor perturbations local to the active site. DHFR-CaM contains five tryptophans in the DFHR domain allowing for the use of tryptophan fluorescence to monitor the unfolding of the DHFR domain. Finally, circular dichroism was used to monitor the unfolding of the entire protein. Since calmodulin is a calcium binding protein the unfolding studies were done both in the presence of EDTA and the presence of calcium. This was done to investigate the effects of structural changes associated with the binding of calcium to calmodulin upon the stability of the DHFR domain. Wild type and mutant DHFRs were compared using AG values determined from equilibrium unfolding curves.

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