Vuister G W, Delaglio F, 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 Jan;3(1):67-80. doi: 10.1007/BF00242476.
Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (+/- 0.5 Hz) measurement of one bond JC alpha H alpha coupling constants in proteins that are uniformly enriched with 13C. An empirical phi,psi-surface is calculated which describes the deviation of 1JC alpha H alpha from its random coil value, using 203 1JC alpha H alpha values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which phi and psi are known with good precision from previous X-ray crystallographic studies. Residues in alpha-helical conformation exhibit positive deviations of 4-5 Hz, whereas deviations in beta-sheet are small and, on average, slightly negative. Data indicate that 1JC alpha H alpha depends primarily on psi, and that 1JC alpha H alpha may be useful as a qualitative probe for secondary structure. Comparison of 1JC alpha H alpha coupling constants measured in free calmodulin and in its complex with a 26-amino-acid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the 'central helix' is even more extensive than in free calmodulin.
描述了对恒时HSQC和HCACO实验的简单伪三维修改，这些修改允许在均匀富集13C的蛋白质中精确测量（±0.5 Hz）一键JCαHα耦合常数。计算了一个经验性的φ，ψ表面，该表面使用为钙调蛋白、葡萄球菌核酸酶和碱性胰蛋白酶抑制剂中的残基测量的203个1JCαHα值描述了1JCαHα与其随机卷曲值的偏差，对于这些蛋白质，先前的X射线晶体学研究已精确得知其φ和ψ值。处于α螺旋构象的残基表现出4 - 5 Hz的正偏差，而β折叠中的偏差较小，平均而言略为负偏差。数据表明1JCαHα主要取决于ψ，并且1JCαHα可用作二级结构的定性探针。对游离钙调蛋白及其与肌球蛋白轻链激酶的26个氨基酸肽片段的复合物中测量的1JCαHα耦合常数的比较证实，钙调蛋白在形成复合物时二级结构得以保留，但“中央螺旋”中部的破坏比游离钙调蛋白中更为广泛。
