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
- 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), Vol. 29:17; ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
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PHENYLALANINE
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550601* - Medicine- Unsealed Radionuclides in Diagnostics