Solution studies of staphylococcal nuclease H124L. 2. sup 1 H, sup 13 C, and sup 15 N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3 prime ,5 prime -bisphosphate-calcium ternary complex

Accurate {sup 1}H, {sup 15}N, and {sup 13}C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone {sup 1}H and {sup 15}N assignments, obtained by analysis of three-dimensional {sup 1}H-{sup 15}N HMQC-NOESY data were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear {sup 1}H NMR studies were at natural isotopic abundance or labeled randomly with {sup 2}H nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with {sup 15}N or uniformly with {sup 13}C. Additional nuclease H124L samples were labeled selectively by incorporating single {sup 15}N- or {sup 13}C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L{center dot}pdTp{center dot}Ca{sup 2+} ternary complex. The results reveal that the binding of pdTp and Ca{sup 2+} induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca{sup 2+}; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.