Studies of hydrogen exchange in proteins. VI. Urea effects on ribonuclease exchange kinetics leading to a general model for hydrogen exchange from folded proteins.
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TL;DR
A hydrogen exchange model in which the low E*app exchange takes place from folded RNase and the high E-app exchange take place from thermally unfolded RNase accounts for the temperature, pH, urea, and ethanol dependence of RNase hydrogen exchange kinetics.
Abstract
Abstract The urea dependence of RNase hydrogen exchange kinetics has been studied. Exchange having apparent low activation energy (≃22 kcal) and exchange having apparent high activation energy (≃60 kcal) were measured separately as a function of urea concentration. The urea dependence of the low apparent activation energy (E*app) exchange is qualitatively different from that with high E*app whether one varies the temperature at constant urea concentration or the urea concentration at constant temperature. The increase in the high E* exchange rate in 2 m urea is quantitatively consistent with a mechanism involving thermal unfolding. The urea effect on the low E*app exchange rate shows a urea concentration dependence identical with that of the urea-induced unfolding. A hydrogen exchange model in which the low E*app exchange takes place from folded RNase and the high E*app exchange takes place from thermally unfolded RNase accounts for the temperature, pH, urea, and ethanol dependence of RNase hydrogen exchange kinetics. This model significantly alters some of the prevalent interpretations of protein hydrogen exchange kinetics. A conformational model of folded RNase must explain the following hydrogen exchange phenomena. (a) Apparently all labile hydrogen sites in a folded protein may exchange without the protein unfolding. (b) The pH dependence of exchange from folded RNase is 4 to 6 times less than in the random conformation. (c) The rank order of exchange from folded RNase is temperature and pH independent. (d) The distribution of exchange rates in folded RNase is over 75-fold wider than those of random conformation RNase.