Conformational stability of ribonuclease T1 determined by hydrogen-deuterium exchange.
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abstract
The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase Tl havo been monitored at 25, 40, 45 and 50 C at pD 5.6 and at 40 and 45 C at pD 6.6. The hydrogen exchange rate constants of the hydrogen bonded residues varied over 8 orders of magnitude at 25 C with thirteen residues showing exchange rates consistent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central 3pleated sheet. The residues located in the o-helix and the remaining strands of the J sheet exhibited exchange behaviors consistent with exchange occurring due to local structural fluctuations. For several residues at 25 C, the global free energy change calculated from the hydrogen exchange data was over 2 kcal / mol greater than the free energy of unfolding determined from urea denaturation experiments. The number of residues showing this unexpected behavior was found to increase with temperature. This apparent inconsistency can be explained quantitatively on the basis of the cis-trans isomerization equilibrium of the two Carolines, Pro 39 and Pro 55 [Mayr, et al., Biochemistry ( 1996) 35, 5550-5561], if the most slowly exchanging hydrogens exchange from a globally higher free energy unfolded state in which both Pro 39 and Pro 55 are still c;,<. as they are in the folded protein.
