Measurement of two- and three-bond 13C-1H J couplings to the C delta carbons of leucine residues in staphylococcal nuclease.
A new 1H-detected 3D NMR experiment is described that permits quantitative measurement of two- and three-bond 13C-1H couplings in proteins with selectively 13C-enriched methyl sites. The method is demonstrated for staphylococcal nuclease selectively [5,5 13C]-labeled in all 11 leucine positions and ligated with thymidine 3',5'-biphosphate and Ca2+. Two- and three-bond 13C methyl-proton couplings are reported and, together with the measured three-bond JC alpha C delta in uniformly 13C-enriched staphylococcal nuclease, the chi 2-angles and the stereospecific assignments of the C delta methyl group with respect to the prochiral beta-protons were determined. The same residues that were previously found to have high degrees of internal mobility on the basis of 13C relaxation times have measured coupling constants that are indicative of motional averaging.
Vuister
G W
GW
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
Yamazaki
T
T
Torchia
D A
DA
Bax
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002247:Carbon Isotopes; D006859:Hydrogen; D007930:Leucine; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008433:Mathematics; D008836:Micrococcal Nuclease / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013942:Thymine Nucleotides / Q000378:metabolism
D002247:Carbon Isotopes; D013942:Thymine Nucleotides; C019111:thymidine 3',5'-diphosphate; D006859:Hydrogen; D008836:Micrococcal Nuclease; D007930:Leucine; D002118:Calcium