Solution Studies of Staphylococcal Nuclease H124L. 2. ¹H, ¹³C, and ¹⁵N Chemical Shift Assignments for the Unligated Enzyme and Analysis of Chemical Shift Changes That Accompany Formation of the Nuclease-Thymidine 3ʹ5ʹ-Bisphosphate-Calcium Ternary Complex

Accurate¹H, ¹⁵N, and ¹³C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone¹H and ¹⁵N assignments, obtained by analysis of three-dimensional¹H-¹⁵N 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 homonuclear¹H NMR studies were at natural isotopic abundance or labeled randomly with ²H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with ¹⁵N (to an isotope level >95%) or uniformly with ¹³C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single ¹⁵N-or ¹³C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124LºpdTpºCa²⁺ 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.