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

Jinfeng Wang, Andrew P. Hinck, Stewart N. Loh, John L. Markley, David M. LeMaster

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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.

Original languageEnglish (US)
Pages (from-to)921-936
Number of pages16
Issue number3
StatePublished - Feb 1 1992
Externally publishedYes


ASJC Scopus subject areas

  • Biochemistry

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