Hydrogen exchange in ribonuclease A and ribonuclease S: Evidence for residual structure in the unfolded state under native conditions

Two-dimensional NMR spectroscopy has been used to monitor the exchange of b ackbone amide protons in ribonuclease A (RNase A) and its subtilisin-cleave d form, ribonuclease S (RNase S). Exchange measurements at two different pH values (5.4 and 6.0) show that the exchange process occurs according to th e conditions of the EX2 limit. Differential scanning calorimetry measuremen ts have been carried out in 2H(2)O under conditions analogous to those used in the NMR experiments in order to determine the values of Delta C-p, Delt a H-u and T-m, corresponding to the thermal denaturation of both proteins. For the amide protons of a large number of residues in RNase A, the free en ergies at 25 degrees C for exchange competent unfolding processes are much lower than the calorimetric denaturation free energies, thus showing that e xchange occurs through local fluctuations in the native state. For 20 other protons, the cleavage reaction had approximately the same effect on the ex change rate constants than on the equilibrium constant for unfolding, indic ating that those protons exchange by global unfolding. There is a good agre ement between the residues to which these protons belong and those involved in the putative folding nucleation site identified by quench-flow NMR stud ies. The unfolding free energies of the slowest exchanging protons, Delta G (ex), as evaluated from exchange data, are much larger than the calorimetri c free energies of unfolding, Delta G(u). Given the agreement between Delta Delta G(ex)((A-S)), the difference in free energy from exchange for a give n proton of the two proteins, and Delta Delta G(u)((A-S)), the difference i n the calorimetric free energy of the two proteins, the discrepancy indicat es that the intrinsic exchange rates in the unfolded state of those protons cannot be approximated by those measured in short unstructured peptides an d, consequently, exchange for those protons in RNase A and S must occur thr ough a rather structured denatured state. (C) 1999 Academic Press.