HYDROGEN-EXCHANGE IN UNLIGATED AND LIGATED STAPHYLOCOCCAL NUCLEASE

The exchange kinetics of over 70% of the 143 backbone amide hydrogens in staphylococcal nuclease H124L (nuclease H124L), both in its unligated state and in its ternary complex with Ca2+ and thymidine 3',5'-bisphosphate, have been quantified by nitrogen-15 resolved proton nuclear magnetic resonance spectroscopy. Protection factors for the slowly exchanging hydrogens in unligated nuclease H124L at 37-degrees-C and pH* 5.5 were found to vary by over one order of magnitude. This range of protection factors has been interpreted in the framework of global and local structural fluctuations. The three most highly protected hydrogens (K24, L25, M26) map to strand 2 of the central five-stranded beta-barrel. The free energy change for the opening reaction which exposes these hydrogens to the solvent (DELTAG-degrees op) was calculated from the exchange rates in the native and denatured states, the latter values being estimated from model peptide exchange studies [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. Close agreement was found between DELTAG-degrees op and DELTAG-degrees u, the free energy change of unfolding as measured by urea denaturation experiments. Exchange of these hydrogens thus appears to occur via global unfolding of the protein. One region exhibited somewhat lower protection factors: it mapped to the C-terminal portions of helix 2 and helix 3 and to part of the intervening segment. This region has been identified as a minor hydrophobic domain of nuclease [Shortle, D., Stites, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041]. The decreased protection factors in this region appear to arise from local structural fluctuations that accompany cis half arrow right over half arrow left trans isomerization about the K116-P117 peptide bond. Inhibitor binding was found to produce global increases in protection factors. The baseline stability increase afforded by inhibitor binding estimated from NH exchange data (DELTADELTAG-degrees op) was found again to be similar to the DELTADELTAG-degrees u value determined from urea unfolding experiments except for in the region affected by the cis half arrow right over half arrow left trans isomerization of the K116-P117 peptide bond. This region showed additional protection attributed to inhibitor-induced perturbation of the cis half arrow right over half arrow left trans equilibrium to the more exchange stable cis conformation. The results of the present study demonstrate that hydrogen exchange kinetics can be used to estimate the global stability of nuclease H124L in the presence and absence of ligands and to pinpoint local changes in structural free energy. In addition, the exchange rates from the unfolded state appear to be well described by the random-coil values. The results provide no evidence for the existence of any stable hydrogen-bonded structure in the denatured state-as reported by residues L24, K25, or M26-under the conditions of the experiment.