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Individual Amide Proton Exchange Rates in Thermally Unfolded Basic Pancreatic Trypsin Inhibitor? Heinrich Roder,* Gerhard Wagner,* and Kurt Wuthrich Institut fur Molekularbiologie und Biophysik, Eidgenossische Technische Hochschule, Zurich- Honggerberg, CH-8093 Zurich, Switzerland Received December 20, 1984 ABSTRACT: A novel experiment is described for measurements of amide proton exchange rates in proteins with a time resolution of about 1 s. A flow apparatus was used to expose protein solutions in 2H20 first to high temperature for a predetermined time period, during which 1H-2H exchange proceeded, and then to ice-water. The technique was applied for exchange studies in thermally unfolded, selectively reduced basic pancreatic trypsin inhibitor. Measurements were made by ‘H nuclear magnetic resonance after the exchange was quenched by rapid cooling. Thereby, the sequence-specific resonance assignments for the folded protein could be used, which had been previously obtained. The results of this study indicate that the exchange rates in the thermally unfolded protein are close to those expected for a random chain and that the N H exchange is catalyzed by 2H+ and 02H- up to high temperature, with no significant contributions from p2H-independent catalysis. We conclude that the parameters derived by Molday et al. [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-1581 from measurements with small model peptides can be used to calculate intrinsic exchange rates in unfolded proteins and thus provide a reliable reference for the interpretation of exchange rates measured under native conditions. M e a s u r e m e n t s of exchange rates of interior labile protons are a widely used method for investigating internal motility in globular proteins (Linderstrram-Lang, 1955; Hvidt & Nielsen, 1966; Englander et al., 1972; Wagner & Wuthrich, 1979a). Usually the data are evaluated on the basis of a structural unfolding model: zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA k k3 k N(H) O(H) - O(2H) N(*H) (1) 2 ’H’O ki This work was supported by the Swiss National Science Foundation (Projects 3.528.79 and 3.284.82). Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA t Present address: 0006-2960/85/0424-7407$01.50/0 N indicates the ensemble of “closed” conformers in which the labile proton considered is not accessible to the deuterated solvent (for example, because of internal hydrogen bonding). 0 indicates the “open” conformers in which the same proton is in contact with the solvent. k, and k2 describe the rates of interchange between closed and open states of the protein, and k3 is the intrinsic exchange rate for the solvent-accessible, labile proton. Depending on the ratios of these rate constants, different limiting kinetic situations may arise (Hvidt & Nielsen, 1966). However, the experimental observations presented in the preceding paper (Roder et al., 1985) indicate that for studies of the exchange of interior backbone amide protons in proteins, exchange via an EX, mechanism will zyxwvutsr 62 1985 American Chemical Society