DETAILED CHARACTERIZATION OF OVOMUCOID THIRD DOMAINS AND OF THEIR INTERACTION WITH ENZYMES

TIAO-YIN LIN, Purdue University

Abstract

Our laboratory is working on a sequence to reactivity algorithm for protein inhibitors of serine proteinases in general and for avian ovomucoid third domains in particular. The algorithm is a set of rules for determining the enzyme-inhibitor association equilibrium constants, K(,a)('obs), from the amino acid sequence. To continue this research we need many purified third domain variants of specified amino acid sequences. Therefore, sensitive techniques for separating variants and methods for generating specified new variants are desired. This thesis addresses both of these problems. A highly sensitive HPLC technique was developed to separate pairs of ovomucoid third domains with closely related sequences. Even when two third domains differ at a single uncharged residue, such as Ser/Gly, they were separated to the base line by reverse phase HPLC on (mu)Bondapak C(,18) columns with a binary gradient including H(,2)O, trifluoroacetic acid and organic solvent. The detector was operated at 214nm. Separations of protein mixtures in this system can be achieved with loads of 1.6(mu)g to 1mg. Proteins differing in length of the chain by one or two (out of 56) neutral amino acids were also resolved. The semisynthetic method has been used to obtain ovomucoid third domain variants with chymotrypsin inhibitory specificity (Wieczorek and Laskowski, 1983). It was applied here to semisynthesize variants with trypsin inhibitor activity. In order to test specific parts of the algorithm, two hybrid third domains that have Lys at reactive site and differ only by a single Gly/Asp change at P(,14)' position were generated. The final yield of the semisynthetic protein was approximately 11%. The P(,14') Gly/Asp change has no effect on Ka('obs)'s of the hybrids with (beta)-trypsin or Streptomyces griseus proteinase B (SGPB). However, the inhibitor with P(,14') Gly is 7 fold stronger for Streptomyces griseus proteinase A (SGPA). The association constants of the hybrids show a P(,1), P(,14') minor non-additivity for SGPA and a P(,1), P(,14'), major nonadditivity for SGPB. Hypotheses explaining these two non-additivities in detailed molecular terms are proposed.

Degree

Ph.D.

Subject Area

Biochemistry

COinS