NUCLEAR MAGNETIC RESONANCE STUDIES OF AVIAN OVOMUCOIDS AND THEIR ISOLATED STRUCTURAL DOMAINS (NMR)
Abstract
Several avian ovomucoids, members of the Kazal family of serine proteinase inhibitors, have been studied by ('1)H and ('13)C NMR spectroscopy. The pH titration properties and deuterium exchange kinetics of the histidine residues were determined in intact chicken ovomucoid, which is composed of three tandem homologous domains, and in isolated structural domains obtained by limited chemical or enzymatic cleavage. The results establish that the properties of the histidines stem primarily from intradomain interactions; however, an interdomain interaction is required to explain the behavior of histidine-182 in the carboxyl terminal (third) domain of the molecule. Investigation of chicken ovomucoid third domain has shown that the low pH transition (pH('*) mid (TURN)2.6) which affects tyrosine-31 of the domain is limited in its conformational consequences. The titration behavior of tyrosine-31 is consistent with its acting as a hydrogen bond donor to an acidic side chain. Evidence was also obtained for an interaction between the imidazole side chain of histidine-52 and the carboxyl group of the carboxy terminal cysteine of the domain. Extension of these studies to third domains from Japanese quail and silver pheasant ovomucoids show that the conformations of the homologous domains are similar. However, limited amino acid replacements between species affect the titration behavior and mobilities of aromatic side chains of the domain. Comparison of ('1)H and ('13)C NMR spectra of glycosylated and non-glycosylated chicken ovomucoid third domain revealed no differences in their conformations. The histidine-57 C(,(epsilon))-H resonance of bovine chymotrypsin A(,(alpha)) and chymotrypsinogen A is altered upon complex formation with silver pheasant third domain; whereas that of histidine-40 is not. The results confirm the assignments of the histidine-57 C(,(epsilon))-H resonances in free bovine chymotrypsin A and chymotrypsinogen A (Markley and Ibanez, 1978). Unfortunatedly the resonance corresponding to histidine-57 C(,(epsilon))-H could not be resolved in either complex. The histidine-52 C(,(epsilon))-H resonance of silver pheasant third domain is unaltered by complex formation with the enzyme or zymogen. In ('2)H(,2)O a lowfield resonance whose chemical shift (15.2 ppm) was independent of pH('*) was observed in complexes of the domain with both the enzyme and zymogen.
Degree
Ph.D.
Subject Area
Nuclear chemistry
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