Two-Dimensional NMR Studies of Staphylococcal Nuclease. 2. Sequence-Specific Assignments of Carbon-13 and Nitrogen-15 Signals from 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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Abstract

Samples of staphylococcal nuclease H124L (cloned protein overproduced in Escherichia coli whose sequence is identical with that of the nuclease isolated from the V8 strain of Staphylococcus aureus) were labeled uniformly with carbon-13 (26% ul 13C), uniformly with nitrogen-15 (95% ul 15N), and specifically by incorporating nitrogen-15-labeled leucine ([98% 15N]Leu) or carbon-13-labeled lysine ([26% ul 13C]Lys), arginine ([26% ul 13C]Arg), or methionine ([26% ul 13C]Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca2+) at pH 5.1 (H20) or pH* 5.5 (2H20) at 45 °C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments at spectrometer fields of 14.09 and 11.74 T: 1H-13C single-bond correlation (1H{13C}SBC); 1H-I3C single-bond correlation with NOE relay (1H{C}SBC-NOE); 1H-13C single-bond correlation with Hartmann-Hahn relay (1H-{13C}SBC-HH); 1H-13C multiple-bond correlation (1H{13C}MBC); 1H-15N single-bond correlation (lH-{15N}SBC); 1H-15N single-bond correlation with NOE relay (’H{15N}SBC-NOE). The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE’s) characteristic of antiparallel β-sheet (daa NOE’s) were observed in the 1H{13C}-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with 13C. NOE's characteristic of a-helix (dNN NOE’s) were observed in the 1H{15N}SBC-NOE spectrum of the complex prepared from protein labeled uniformly with 15N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on 1H{1H} 2D NMR experiments [Wang, J., LeMaster, D. M., & Markley, J. L. (1990) Biochemistry (preceding paper in this issue)].

Original languageEnglish (US)
Pages (from-to)102-113
Number of pages12
JournalBiochemistry
Volume29
Issue number1
DOIs
StatePublished - Jan 1 1990
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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