Two-dimensional NMR studies of staphylococcal nuclease. 2. Sequence-specific assignments of carbon-13 and nitrogen-15 signals from the nuclease H124L-thymidine 3 prime ,5 prime -bisphosphate-Ca sup 2+ ternary complex
- Univ. of Wisconsin, Madison (USA)
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 {sup 13}C), uniformly with nitrogen-15 (95% ul {sup 15}N), and specifically by incorporating nitrogen-15-labeled leucine ((98% {sup 15}N)Leu) or carbon-13-labeled lysine ((26% ul {sup 13}C)Lys), arginine ((26% ul {sup 13}C)Arg), or methionine ((26% ul {sup 13}C)Met). Solutions of the ternary complexes of these analogues (nuclease H124L-pdTp-Ca{sup 2+}) at pH 5.1 (H{sub 2}O) or pH 5.5 ({sup 2}H{sub 2}O) at 45{degree}C were analyzed as appropriate to the labeling pattern by multinuclear two-dimensional (2D) NMR experiments spectrometer fields of 14.09 and 11.74 T. The results have assisted in spin system assignments and in identification of secondary structural elements. Nuclear Overhauser enhancements (NOE's) characteristic of antiparellel {beta}-sheet (d{sub {alpha}{alpha} }NOE's) were observed in the {sup 1}H({sup 13}C)-SBC-NOE spectrum of the nuclease ternary complex labeled uniformly with {sup 13}C. NOE's characteristic of {alpha}-helix were observed in the {sup 1}H({sup 15}N)SBC-NOE spectrum of the complex prepared from protein labeled uniformly with {sup 15}N. The assignments obtained from these multinuclear NMR studies have confirmed and extended assignments based on {sup 1}H({sup 1}H) 2D NMR experiments.
- OSTI ID:
- 6827583
- Journal Information:
- Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:1; ISSN 0006-2960; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Solution studies of staphylococcal nuclease H124L. 2. sup 1 H, sup 13 C, and sup 15 N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3 prime ,5 prime -bisphosphate-calcium ternary complex
Two-dimensional NMR studies of staphylococcal nuclease. 1. Sequence-specific assignments of hydrogen-1 signals and solution structure of the nuclease H124L-thymidine 3 prime ,5 prime -bisphosphate-Ca sup 2+ ternary complex
Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
ALKALINE EARTH METAL COMPLEXES
ALKALINE EARTH METAL COMPOUNDS
AMINO ACID SEQUENCE
AMINO ACIDS
AZINES
BACTERIA
BARYONS
CALCIUM COMPLEXES
CALCIUM COMPOUNDS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
CATIONS
CHARGED PARTICLES
COMPLEXES
DEUTERIUM COMPOUNDS
ELEMENTARY PARTICLES
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
FERMIONS
HADRONS
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
HYDROLASES
IONS
ISOTOPES
LEUCINE
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEASES
NUCLEI
NUCLEONS
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OVERHAUSER EFFECT
PHOSPHODIESTERASES
PROTONS
PYRIMIDINES
RESONANCE
RIBOSIDES
STABLE ISOTOPES
STAPHYLOCOCCUS
THYMIDINE