The Influence of a Single Salt Bridge on Static and Dynamic Features of the Globular Solution Conformation of the Basic Pancreatic Trypsin Inhibitor. 1H and 13C Nuclear-Magnetic-Resonance Studies of the Native and the Transaminated Inhibitor

Abstract

Evidence for a salt bridge interaction between the amino terminus and the carboxyl terminus in the basic pancreatic trypsin inhibitor was obtained from the pH titration curves of the terminal amino acid residues determined by 1H and 13C nuclear magnetic resonance (NMR). This interpretation of the titration behavior was confirmed by studies of an inhibitor modified by transamination of the α-amino group of Arg-1. The NMR techniques thus revealed a local difference between the protein surface structures in the crystal and in solution. Furthermore, the influence of the salt bridge between the end groups on the protein conformation was investigated by comparative studies of the native and the transminated inhibitor. In all, over 200 corresponding NMR spectral and structural parameters in the two species were considered. The molecular conformations of the native and the transaminated inhibitor were found to be nearly identical, with small differences strictly localized in the immediate spatial environment of the chain termini. However, the absence of the salt bridge in the transaminated inhibitor affected the overall stability of the globular protein conformation, as evidenced in a lower denaturation temperature and an increased rate of exchange of interior amide protons with the solvent. The contribution of the single salt bridge between the chain termini to the stability of the globular protein conformation was estimated to be approximately 4.2 kJ mol-1.