Two-Dimensional NMR Studies of Staphylococcal Nuclease: Evidence for Conformational Heterogeneity from Hydrogen-1, Carbon-13, and Nitrogen-15 Spin System Assignments of the Aromatic Amino Acids in the Nuclease H124L-Thymidine 3′,5′-Bisphosphate-Ca2+ Ternary Complex

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

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A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined 1H, 13C, and 15N spin systems of the single tryptophan, three phenylalanines, three histidines, and seven tyrosines of staphylococcal nuclease H124L in its ternary complex with calcium and thymidine 3′,5′-bisphosphate at pH 5.1 (H2O) or pH* 5.5 (2H2O). Samples of recombinant nuclease were labeled with 13C or 15N as appropriate to individual NMR experiments: uniformly with 15N (all sites to >95%), uniformly with 13C (all sites to 26%), selectively with 13C (single amino acids uniformly labeled to 26%), or selectively with 15N (single amino acids uniformly labeled to >95%). NMR data used in the analysis included single-bond and multiple-bond 1H-13C and multiple-bond 1H-15N correlations, 1H-13C single-bond correlation with Hartmann-Hahn relay (1H{13C}SBC-HH), and 1H-13C single-bond correlation with NOE relay (1H{13C}SBC-NOE). The aromatic protons of the spin systems were identified from 1H{13C)SBC-HH data, and the nonprotonated aromatic ring carbons were identified from 1H-13C multiple-bond correlations. Sequence-specific assignments were made on the basis of observed NOE relay connectivities between assigned 1Hα-l3Cα or 1Hβ-13Cβ direct cross peaks in the aliphatic region [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry 29, 88–101] and 1Hδ-13Cδ direct cross peaks in the aromatic region of the 1H{13C}SBC-NOE spectrum. The His121 1Hδ2 resonance, which has an unusual upfield shift (at 4.3 ppm in the aliphatic region), was assigned from 1H{13C}SBC, 1H{13C}MBC, and 1H{15N}MBC data. Evidence for local structural heterogeneity in the ternary complex was provided by doubled peaks assigned to His46, one tyrosine, and one phenylalanine. Measurement of NOE buildup rates between protons on different aromatic residues of the major ternary complex species yielded a number of interproton distances that could be compared with those from X-ray structures of the wild-type nuclease ternary complex with calcium and thymidine 3′,5′-bisphosphate [Cotton, F. A., Hazen, E. E., Jr., & Legg, M. J. (1979) Proe. Natl. Acad. Sci. U.S.A. 76, 2551–2555; Loll, P. J., & Lattman, E. E. (1989) Proteins: Struct., Funct., Genet. 5, 183–201]. The unusual chemical shift of His121 1Hδ2 is consistent with ring current calculations from either X-ray structure.

Original languageEnglish (US)
Pages (from-to)4242-4253
Number of pages12
Issue number17
Publication statusPublished - Mar 1 1990


ASJC Scopus subject areas

  • Biochemistry

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