Two-dimensional NMR studies of staphylococcal nuclease: Evidence for conformational heterogeneity from hydrogen-1, carbon-13, and nitrogen-15 spin systems assignments of the aromatic amino acids in the nuclease H124L-thymidine 3 prime ,5 prime -bisphosphate-Ca sup 2+ ternary complex

Journal Article · Tue May 01 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00469a029· OSTI ID:6071042

Wang, J; Hinck, A P; Loh, S N; Markley, J L ^[1]

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1. Univ. of Wisconsin, Madison (USA)

A combination of multinuclear two-dimensional NMR experiments served to identify and assign the combined {sup 1}H, {sup 13}C, and {sup 15}N 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{prime},5{prime}-bisphosphate at pH 5.1 (H{sub 2}O) or pH* 5.5 ({sup 2}H{sub 2}O). Samples of recombinant nuclease were labeled with {sup 13}C or {sup 15}N as appropriate to individual NMR experiments: uniformly with {sup 15}N (all sites to >95%), uniformly with {sup 13}C (all sites to 26%), selectively with {sup 13}C (single amino acids uniformly labeled to >26%), or selectively with {sup 15}N (single amino acids uniformly labeled to >95%). NMR data used in the analysis included single-bond and multiple-bond {sup 1}H-{sup 13}C and multiple-bond {sup 1}H-{sup 15}N correlations, {sup 1}H-{sup 13}C single-bond correlations with Hartmann-Hahn relay ({sup 1}H({sup 13}C)SBC-HH), and {sup 1}H-{sup 13}C single-bond correlation with NOE relay ({sup 1}H({sup 13}C)SBC-NOE). The aromatic protons of the spin systems were identified from {sup 1}H({sup 13}C)SBC-HH data, and the nonprotonated aromatic ring carbons were identified from {sup 1}H-{sup 13}C multiple-bond correlations. 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{prime},5{prime}-bisphosphate.

• OSTI ID: 6071042
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:17; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ALKALINE EARTH METAL COMPOUNDS
AMINO ACIDS
AROMATICS
AZAARENES
AZINES
AZOLES
BACTERIA
CALCIUM COMPOUNDS
CARBON 13
CARBON ISOTOPES
CARBOXYLIC ACIDS
ENZYMES
ESTERASES
EVEN-ODD NUCLEI
HEAVY WATER
HETEROCYCLIC ACIDS
HETEROCYCLIC COMPOUNDS
HISTIDINE
HYDROGEN 1
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
HYDROLASES
HYDROXY ACIDS
IMIDAZOLES
INDOLES
ISOTOPES
LIGHT NUCLEI
MAGNETIC RESONANCE
MICROORGANISMS
MOLECULAR STRUCTURE
NITROGEN 15
NITROGEN ISOTOPES
NUCLEAR MAGNETIC RESONANCE
NUCLEASES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-EVEN NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXYGEN COMPOUNDS
PHENYLALANINE
PHOSPHODIESTERASES
PYRIMIDINES
PYRROLES
RESONANCE
RIBOSIDES
STABLE ISOTOPES
STAPHYLOCOCCUS
THYMIDINE
TRYPTOPHAN
TYROSINE
WATER