SINGLE PROTON EXCHANGE KINETICS IN PROTEINS

这个项目的总体目标是确定的内部运动

The general goal of this project is to determine the internal motions of proteins by which atoms buried in the matrix of folded proteins are accessible to solvent. This is measured by the hydrogen isotope exchange kinetics of solvent hydrogen atoms with labile protein protons, most of which are peptide amide protons. The hydrogen isotope exchange kinetics of peptide NN's in folded proteins are distributed over 6-10 orders of magnitude. The most rapidly exchangeing NH's are on the surface, and the slowest exchanging NH's tend to be in buried sections of Beta -sheet. Although buried NH's exchange slower than model compounds, their finite exchange rates demonstrate that interior atoms in native proteins have some probability of being exposed to solvent. This immediately implies that the protein fluctuates to render tightly packed, buried atoms accessible to solvent. Recently our laboratory has made the exciting findings that the exchange rates of NH's on the surface of bovine pancreatic trypsin inhibitor (BPTI) vary from 3-fold to 2000-fold slower than model compounds, that their pHmin values vary over greater than 2 pH units, and that some have pHmin of less than 1. It has been commonly assumed that surface NH atoms (not H-bonded and with finite static accessibility) exchange with rates comparable to those in model peptides. The deviation in exchange rates and pHmin from model compounds then taken on special interest as a reflection of the dynamic structure of the protein-solvent interface. The specific aims of this research proposal are 1) to characterize the hydrogen exchange kinetics of NH's in BPTI with intermediate exchange rates, 2) to measure the effect of trypsinogen and trypsinogen/Ile-Val binding on the exchange rates of BPTI NH's 3) to compare the exchange rate of individual BPTI NH's in solution with their exchange rates in the crystalline and powder forms, and 4) to measure the exchange kinetics of water buried in the BPTI-trypsin complex with solvent water.