Effect of trypsin binding on the hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor .beta.-sheet NH's

Abstract

The hydrogen-deuterium exchange rates of the slowest exchanging @-sheet NH’s in the bovine pancreatic trypsin inhibitor (BPTI) have been determined in free BPTI and in the trypsin-BPTI complex at pH 9-10,2540 OC. Rate constants for individual protons have been measured from their assigned resonances in the ‘H NMR spectrum. Trypsin binding has a highly localized effect; the Tyr-35 N H exchange x e exchange of isotope between water hydrogens and labile protein hydrogens is a measure of the accessibility of solvent to the protein’s internal sites. The largest group of exchangeable protons in proteins are peptide NH’s. Exchange rate constants of NH’s in extended polypeptides at pH 3 and 25 OC are around IO-’ min-’, while exchange rate constants of NH’s in folded proteins under the same conditions vary from about lo-’ to min-I. Although labile protons in folded From the Department of Biochemistry, University of Minnesota, St. Paul, Minnesota 55 108. Received September 23, 1983. Supported by Grants NSF PCM8101993 and NIH GM26242. Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1502 Budapest, Hungary. *Present address: rate is slowed by a factor of > lo3 in the complex, while the other NH’s measured are slowed by a factor of 3-15. In free BPTI, under conditions where the exchange rate constants have activation energies in the range 11-32 kcal/mol, the NH’s of Tyr-21, Phe-22, and Tyr-23 are several orders of magnitude slower than the other ,s Englander et al., 1972; Woodward & Hilton, 1979; Woodward et al., 1982; Barksdale & Rosenberg, 1982). Traditionally, measurements of protein hydrogen exchange kinetics average over all exchangeable protons (Englander & Englander, 1978). More recently, assignments of average NH exchange rates to segments of the protein sequence have been made by rapid proteolysis methods (Rosa & Richards, 1981; Englander et al., 1980). Now, with the development of procedures for assigning NMR N H resonances (Dubs et al., 1979; Delpierre et al., 1982; Kuwajima & Baldwin, 1983), it is 0006-2960/84/0423-2064$01 .SO10