CONFORMATIONAL STABILITY OF RIBONUCLEASE TL DETERMINED BY HYDROGEN-DEUTERIUM EXCHANGE

The hydrogen-deuterium exchange kinetics of 37 backbone amide residues in RNase T1 have been monitored at 25, 40, 45, and 50 degrees C at pD 5.6 and at 40 and 45 degrees C at pD 6.6. The hydrogen exchange rate constants of the hydrogen-bonded residues varied over eight orders of magnitude at 25 degrees C with 13 residues showing exchange rates cons istent with exchange occurring as a result of global unfolding. These residues are located in strands 2-4 of the central beta-pleated sheet. The residues located in the alpha-helix and the remaining strands of the beta-sheet exhibited exchange behaviors consistent with exchange o ccurring due to local structural fluctuations. For several residues at 25 degrees C, the global free energy change calculated from the hydro gen 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 quantitative ly if the cis-trans isomerization of the two cis prolines, Pro-39 and Pro-55, is taken into account. The cis-trans isomerization equilibrium calculated from kinetic data indicates the free energy of the unfolde d state will be 2.6 kcal/mol higher at 25 degrees C when the two proli nes are cis rather than trans (Mayr LM, Odefey CO, Schutkowski M, Schm id FX. 1996. Kinetic analysis of the unfolding and refolding of ribonu clease T1 by a stopped-flow double-mixing technique. Biochemistry 35: 5550-5561). The hydrogen exchange results are consistent with the most slowly exchanging hydrogens exchanging from a globally higher free en ergy unfolded state in which Pro-55 and Pro-39 are still predominantly in the cia conformation. When the conformational stabilities determin ed by hydrogen exchange are corrected for the proline isomerization eq uilibrium, the results are in excellent agreement with those from an a nalysis of urea denaturation curves.