Protein internal flexibility and global stability: Effect of urea on hydrogen exchange rates of bovine pancreatic trypsin inhibitor

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Abstract
The hydrogen isotope exchange kinetics of buried NH protons in bovine pancreatic trypsin inhibitor (BPTI) was measured in 8 M urea at 30 degrees C and pH 3.5. The data were analyzed by the two-process model in which slower exchanging protons utilize an unfolding mechanism and more rapidly exchanging protons exchange from the folded state. Urea accelerates the set of protons exchanging by the unfolding mechanism, all of which have approximately the same exchange rate constants in urea. For protons in this set, the ratio of exchange rate constants in the presence and absence of urea is used to estimate delta delta G(0-->8M urea) = 6.6 kcal/mol. For the set of protons exchanging from the folded state, 8 M urea either has no effect or slows exchange. Slowing of exchange by urea implies binding of urea to sites at or near the exchanging proton. Some buried protons exchanging from the folded state have diminished rates in 8 M urea, meaning that urea is accessible to these buried sites. Several unassigned side-chain NH's of arginine or lysine are highly protected from exchange by urea, suggesting that they are the location of urea binding sites on the surface of the molecule.

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