Structural and functional consequences of breaking nonreactive site bonds in protein inhibitors of serine proteinases
Abstract
Most protein inhibitors of serine proteinases are small, highly disulfide bridged proteins with high stability and resistance to proteolysis. They show strong, reversible inhibition of serine proteinases. While the bulk of the three-dimensional structures of inhibitors of these various families is significantly different, the local three-dimensional structure of the region that makes direct contact with enzyme in complex is remarkably similar. In addition, the contact region relies on the bulk of the protein for its unique conformation. The two regions have been named the contact region and the scaffolding region. It appears that the need to constrain the contact region to its unique conformation can be met by a number of "scaffolding" designs. In spite of the promiscuity of this union, the scaffolding of the contact region is clearly required for high activity. Careful design of (1) a peptide inhibitor consisting of just the contact residues and (2) a denatured inhibitor lacking all native disulfide bridges indicates that the scaffolding provides a factor of 10$\sp5$ to 10$\sp7$ to K$\sb{\rm a}$ (the equilibrium association constant). The scaffolding structure of various serine proteinase inhibitors was altered in a carefully devised fashion. The alterations were made by breaking single bonds in the inhibitor, e.g. rupture of a disulfide bridge, chemical cleavage of a single peptide bond. The effects on stability, resistance to proteolysis, secondary structural content and inhibitory activity were then measured. Alterations of the local three-dimensional structure of the contact regions are also discussed. The findings are supported and extended to other families of serine proteinase inhibitors by other single cleavages obtained and analyzed in PSTI, ovomucoid second and third domains, STI and other BPTI variants. (Abstract shortened with permission of author.)
Degree
Ph.D.
Advisors
Laskowski, Purdue University.
Subject Area
Biochemistry
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