SOLUTION STUDIES OF STAPHYLOCOCCAL NUCLEASE H124L .2. H-1, C-13, AND N-15 CHEMICAL-SHIFT ASSIGNMENTS FOR THE UNLIGATED ENZYME AND ANALYSIS OF CHEMICAL-SHIFT CHANGES THAT ACCOMPANY FORMATION OF THE NUCLEASE THYMIDINE 3',5'-BISPHOSPHATE CALCIUM TERNARY COMPLEX

Accurate H-1, N-15, and C-13 chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone H-1 and N-15 assignments, obtained by analysis of three-dimensional H-1-N-15 HMQC-NOESY data [Wang, J., Mooberry, E. S., Walkenhorst, W. F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear H-1 NMR studies were at natural isotopic abundance or labeled randomly with H-2 (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with N-15 (to an isotope level > 95%) or uniformly with C-13 (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single N-15- or C-13-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [ Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ 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 Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.