Vuister G W, Yamazaki T, Torchia D A, Bax A
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.
J Biomol NMR. 1993 May;3(3):297-306. doi: 10.1007/BF00212516.
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.
本文描述了一种新的1H检测的3D NMR实验，该实验能够对具有选择性13C富集甲基位点的蛋白质中的二键和三键13C-1H偶合进行定量测量。该方法已在所有11个亮氨酸位置选择性地用[5,5 13C]标记并与胸苷3',5'-二磷酸和Ca2+连接的葡萄球菌核酸酶中得到验证。报道了二键和三键13C甲基-质子偶合，并结合在均匀13C富集的葡萄球菌核酸酶中测得的三键JCαCδ，确定了χ2角以及Cδ甲基相对于前手性β-质子的立体特异性归属。先前基于13C弛豫时间发现具有高度内部流动性的相同残基，其测得的偶合常数表明存在运动平均效应。
