Protein flexibility and function in bovine pancreatic trypsin inhibitor

Changes in protein flexibility resulting from amino acid replacements and trypsin-binding were studied in bovine pancreatic trypsin inhibitor (BPTI). The effects of enhanced free inhibitor flexibility on the BPTI-trypsin interaction were examined by solving the x-ray structure of the Y35G variant in complex with the protease.; 15N NMR relaxation experiments were conducted on BPTI variants involving single site replacements of residues Tyr23, Phe33, Asn43, and Asn44. The results were compared to those obtained previously for three variants of Tyr35. Of all these replacements, only those at Phe33 and Tyr35 caused appreciable effects on BPTI backbone dynamics. Tyr35 variants displays enhanced flexibility throughout the entire trypsin-binding region of the protein, and the F33L protein displays similar effects, but involving a smaller set of residues. These results suggest that Tyr35 plays a dominant role in defining the proper conformation of the BPTI active site.; In contrast to the free protein, the x-ray structure of trypsin-bound Y35G BPTI showed the inhibitor adopting a well-defined conformation nearly indistinguishable from the wild type. These results indicate that extensive flexibility within the Y35G BPTI backbone is lost upon binding trypsin, accounting for a 3 kcal/mol destabilization of the complex. The structural and dynamic effects are correlated with diminished, but substantial, resistance to proteolysis.; 15N and 2H NMR relaxation experiments showed that wild type BPTI backbone amide and sidechain methyl groups display similar dynamics in both the free and trypsin-bound forms. The results suggest that, in contrast to Y35G BPTI, the wild type protein loses very little conformational entropy upon binding to trypsin.; The combined results presented here have implications for the amino acid determinants of protein flexibility and the idea that conformational rigidity is important for function as a protease inhibitor. Namely, specific regions of a protein can be more susceptible to changes in dynamics resulting from mutations, and the changes in protein flexibility can be highly dependent on the mutation site location. Enhanced flexibility in BPTI leads to destabilization of its complex with trypsin as well as diminished resistance to proteolysis. However, substantial inhibitory function is retained provided there is enough inhibitor-protease complex stability.