Abstract:
:Staphylococcal nuclease H124L is a recombinant protein produced in Escherichia coli whose sequence is identical with that of the nuclease produced by the V8 variant of Staphylococcus aureus. The enzyme-metal ion activator-nucleotide inhibitor ternary complex, nuclease H124L-thymidine 3',5'-bisphosphate-Ca2+, was investigated by two-dimensional (2D) NMR techniques. Efficient overproduction of the enzyme facilitated the production of random fractionally deuterated protein, which proved essential for detailed NMR analysis. 1H NMR spin systems were analyzed by conventional 2D 1H[1H] methods: COSY, relayed COSY, HOHAHA, and NOESY. Assignments obtained by 1H NMR experiments were confirmed and extended by 1H-13C and 1H-15N heteronuclear NMR experiments [Wang, J., Hinck, A. P., Loh, S. N., & Markley, J. L. (1990) Biochemistry (following paper in this issue)]. Spectra of the ternary complexes prepared with protein at natural abundance and at 50% random fractional deuteration provided the information needed for sequence-specific assignments of 121 of the 149 amino acid residues. Short- and intermediate-range NOE connectivities allowed the determination of secondary structural features of the ternary complex: three alpha-helical domains and three antiparallel beta-pleated sheets with several reverse turns. A number of nonsequential long-range HN-HN and H alpha-HN connectivities revealed additional information about the spatial arrangement of these secondary structural elements. The solution structure of this ternary complex shows a close correspondence to the crystal structure of the nuclease wt-thymidine 3',5'-bisphosphate-Ca2+ ternary complex [Cotton, F. A., Hazen, E. E., & Legg, M. J. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 2551-2555].
journal_name
Biochemistryjournal_title
Biochemistryauthors
Wang JF,LeMaster DM,Markley JLdoi
10.1021/bi00453a011subject
Has Abstractpub_date
1990-01-09 00:00:00pages
88-101issue
1eissn
0006-2960issn
1520-4995journal_volume
29pub_type
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