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 unligat ed state and in its ternary complex with Ca2+ and thymidine 3',5'-bisp hosphate, have been quantified by nitrogen-15 resolved proton nuclear magnetic resonance spectroscopy. Protection factors for the slowly exc hanging 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 p rotection factors has been interpreted in the framework of global and local structural fluctuations. The three most highly protected hydroge ns (K24, L25, M26) map to strand 2 of the central five-stranded beta-b arrel. The free energy change for the opening reaction which exposes t hese hydrogens to the solvent (DELTAG-degrees op) was calculated from the exchange rates in the native and denatured states, the latter valu es being estimated from model peptide exchange studies [Molday, R. S., Englander, S. W., & Kallen, R. G. (1972) Biochemistry 11, 150-158]. C lose agreement was found between DELTAG-degrees op and DELTAG-degrees u, the free energy change of unfolding as measured by urea denaturatio n experiments. Exchange of these hydrogens thus appears to occur via g lobal unfolding of the protein. One region exhibited somewhat lower pr otection 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 i dentified as a minor hydrophobic domain of nuclease [Shortle, D., Stit es, W. E., & Meeker, A. K. (1990) Biochemistry 29, 8033-8041]. The dec reased protection factors in this region appear to arise from local st ructural fluctuations that accompany cis half arrow right over half ar row left trans isomerization about the K116-P117 peptide bond. Inhibit or binding was found to produce global increases in protection factors . The baseline stability increase afforded by inhibitor binding estima ted from NH exchange data (DELTADELTAG-degrees op) was found again to be similar to the DELTADELTAG-degrees u value determined from urea unf olding experiments except for in the region affected by the cis half a rrow right over half arrow left trans isomerization of the K116-P117 p eptide bond. This region showed additional protection attributed to in hibitor-induced perturbation of the cis half arrow right over half arr ow 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 H124 L in the presence and absence of ligands and to pinpoint local changes in structural free energy. In addition, the exchange rates from the u nfolded state appear to be well described by the random-coil values. T he results provide no evidence for the existence of any stable hydroge n-bonded structure in the denatured state-as reported by residues L24, K25, or M26-under the conditions of the experiment.