Amide proton exchange and surface conformation of the basic pancreatic trypsin inhibitor in solution: Studies with two-dimensional nuclear magnetic resonance

Abstract

A novel approach for studies of amide proton exchange in proteins is presented. It relies on measurements of the amide proton-C α proton cross-peak intensities in the two-dimensional homonuclear correlated 1H nuclear magnetic resonance spectra. The protein is dissolved in 2H 2O and the solution is exposed to the conditions of p 2H and temperature where the exchange rates are to be measured. After variable intervals, the amide proton exchange in a sample of this protein solution is quenched by lowering the temperature and possibly by p 2H variation, and a COSY † † Abbreviations used: n.m.r., nuclear magnetic resonance; 2D n.m.r., 2-dimensional n.m.r.; COSY, 2D correlated spectroseopy; BPTI, basic pancreatic trypsin inhibitor; p.p.m., parts per million. spectrum of this sample is then recorded. Comparison of the NHC αH cross-peak intensities in the spectra recorded after different exchange times yields exchange rates for the individual amide protons. The main advantage compared to previously described techniques is that a much more complete set of individual amide proton exchange rates can be obtained. In the basic pancreatic trypsin inhibitor, where all the amide proton resonances were previously individually assigned, quantitative exchange rates were obtained for 38 of the total of 53 backbone amide protons, and for 14 additional protons lower limits for the exchange rates were established from comparison of the COSY spectra recorded in H 2O and in 2H 2O. Proton exchange data were thus for the first time obtained for numerous peptide groups that are located near the protein surface in the single crystal structure of BPTI. For some locations on the protein surface, it appears that the amide proton exchange rates cannot be correlated readily with the static accessible surface areas in the crystal structure.