Solvent accessibility in folded proteins. Studies of hydrogen exchange in trypsin

Abstract

In a native protein, the exchange of a peptide amide proton with solvent occurs by one of two pathways, either directly from the folded protein, or via unfolding, exchange taking place from the unfolded protein. From the thermal unfolding rate constants, the contribution of unfolding to the over-all kinetics as a function of solvent and temperature has been determined. Exchange involving unfolding of the protein is characterized by a high activation energy, in the range of 50 to 60 Cal per mol. The activiation energy (Eapp) of the rates of exchange directly from the folded protein is approximately 20 to 25 Cal per mol. Because for the proton transfer step, Eapp approximately equal to 20 Cal per mol, the activation energy for any contributing protein conformational process(es) is approximately equal to 0 to 5 Cal per mol. Most, if not all, of the peptide amide protons in a folded protein can exchange directly with solvent without the protein unfolding. The number of "slowly" exchanging protons at a given condition of pH and temperature is not related to a discrete structural unit, but rather to the distribution of observed rates within the broader distribution of actual rates. The large attenuation of hydrogen exchange rates in folded proteins, resulting in a distribution of first order rates over 6 orders of magnitude, is primarily due to the effects of restricted solvent accessibility of labile protons in the three-dimensional structure. Any protein conformational process, such as protein fluctuations, invoked to explain the solvent accessibility must be of low activation energy and attenuated by ethanol and other co-solvents (Woodward, C. K., Ellis, L. M., and Rosenberg, A. (1974) J. Biol. Chem. 250, 440-444).