Pressure Dependence of Amide Hydrogen−Deuterium Exchange Rates for Individual Sites in T4 Lysozyme

Publisher Website
Google Scholar
Abstract
We report measurements of the pressure dependence of rate constants for the exchange of amide residue protons with solvent deuterium for T4 lysozyme. Data obtained at nine pressures from 0.1 to 200 MPa are analyzed using an elementary kinetic model and the formalism of transition state theory which yield activation volumes for the exchange process. Resolution of individual amide sites was accomplished using the HSQC two-dimensional (2D) NMR experiment on uniformly (15)N-labeled protein. The observed activation volumes span the range from 2.75 to -25.1 mL/mol at 22 degreesC and pH* 7.5. When corrected for the pressure dependence of the ionic product for water and for the reported activation volume for the amide exchange reaction in model compounds, the portion of the activation volume associated with the accessibility of the solvent or catalyst to the amide sites ranges from -15.1 to 12.8 mL/mol. There is no simple correlation between the activation volumes and the protection factors for amide hydrogen exchange. The activation volumes for residues in close proximity in either the primary sequence or the folded structure may differ considerably. There is no trivial correlation between the activation volume and the secondary structural unit in which a residue is located, and activation volumes for residues that are apparently structurally coupled may be very different. The modest sizes of the activation volumes obtained under these conditions are in contrast to large values reported for bovine pancreatic trypsin inhibitor at more extreme conditions of 60 degreesC and pH* 8 where major unfolding events or structural rearrangements may dominate the mechanism [Wagner, G. (1983) Q. Rev. Biophys. 16, 1-57].