Hydrogen-tritium exchange kinetics of soybean trypsin inhibitor (kunitz). Solvent accessibility in the folded conformation

The hydrogen exchange kinetics of Kunitz soybean trypsin inhibitor (STI) has been studied at pH 2, 3, and 6.5. From the temperature dependence of proton exchange at low pH, the contribution of major, reversible protein unfolding to the hydrogen exchange kinetics has been determined. Exchange directly from the folded conformation is characterized by an apparent activation energy (E*_app) of approximately 25 kcal/mol, close to that of the chemical exchange step. At pH 6.5 the protein is more temperature stable than at low pH, and exchange of all but ≃8 protons can be observed to exchange with E*_app ≃ 27 kcal/mol. This implies that all but ≃8 protons are accessible to exchange with solvent in the solution structure of folded STI. Estimates can be made of the average number of water molecules per molecule of STI consistent with a solvent accessibility model of hydrogen exchange kinetics. These estimates indicate that very few water molecules within the protein matrix are necessary to explain the exchange data. Calculations are done for the STI hydrogen exchange kinetics at pH 3, 30°, approximating STI structure by a sphere of radius = 18 Å. These calculations indicate an average of ≃4 water molecules in the shell from 13 to 16 Å from the center of the molecule, while