Semisynthesis of ovomucoid third domain variants: Structure-function studies

Theresa Louise Bigler, Purdue University

Abstract

The purpose of this thesis work has been to aid in the study of protein structure-function relationships. The study of structure-function relationships requires first a reference frame, second the ability to selectively change that frame, and finally the ability to quantitatively measure the effect of that change. The reference frame chosen was the serine protein proteinase inhibitor, turkey ovomucoid third domain (OMTKY3). The method selected and explored for making selective changes to this frame was semisynthesis (Wieczorek and Laskowski, 1983; Wieczorek et al., 1987). The function measured was enzyme-inhibitor association (K$\sb{\rm a}$). In this age of great technological advances in protein chemistry i.e. the advent of site-directed mutagenesis and full synthesis, the question may be asked: is semisynthesis a viable method for the preparation of variants today; is semisynthesis unique and is that uniqueness an advantage? The main advantage of semisynthesis has been its ability to selectively incorporate, into the OMTKY3 sequence, noncoded amino acids at important positions, such as P$\sb1$ (Schechter and Berger, 1967). Another advantage of semisynthesis is that disulfide interchange, the first step in semisynthesis, generates a modified inhibitor. This is unique in that site-directed mutagenesis and complete synthesis do not, at any point, generate a modified variant. Modified variants are essential to k$\sb{\rm on}\sp{*}$ and K$\sb{\rm hyd}$ enzyme-inhibitor studies. The preparation of 23 designed semisynthetic OMTKY3 variants enabled the study of protein structure-function relationships. The size of the S$\sb1$ specificity pocket for bovine $\alpha$-chymotrypsin, subtilisin Carlsberg, human leukocyte elastase porcine pancreatic elastase, Streptomyces griseus proteinase A, Streptomyces griseus proteinase B was probed by variants with noncoded amino acids at P$\sb1$. Additivity was demonstrated. A specific inhibitor of GluSGP was prepared. The existence of the P$\sb2\...$P$\sp\prime\sb1$ H-bond in OMTKY3 was experimentally demonstrated. The k$\sb{\rm on*}$ value for $\alpha$-chymotrypsin with a modified OMTKY3 variant was greatly enhanced by the presence of GlyNH$\sb2$ at position 18A. The effect of $\beta$ and $\gamma$-branching at P$\sb1$ on enzyme-inhibitor association was measured.

Degree

Ph.D.

Advisors

Laskowski, Purdue University.

Subject Area

Biochemistry

COinS