Gardner K H, Rosen M K, Kay L E
Department of Medical Genetics and Microbiology, University of Toronto, Ontario, Canada.
Biochemistry. 1997 Feb 11;36(6):1389-401. doi: 10.1021/bi9624806.
The development of 15N, 13C, 2H multidimensional NMR spectroscopy has facilitated the assignment of backbone and side chain resonances of proteins and protein complexes with molecular masses of over 30 kDa. The success of these methods has been achieved through the production of highly deuterated proteins; replacing carbon-bound protons with deuterons significantly improves the sensitivity of many of the experiments used in chemical shift assignment. Unfortunately, uniform deuteration also radically depletes the number of interproton distance restraints available for structure determination, degrading the quality of the resulting structures. Here we describe an approach for improving the precision and accuracy of global folds determined from highly deuterated proteins through the use of deuterated, selectively methyl-protonated samples. This labeling profile maintains the efficiency of triple-resonance NMR experiments while retaining a sufficient number of protons at locations where they can be used to establish NOE-based contacts between different elements of secondary structure. We evaluate how this deuteration scheme affects the sensitivity and resolution of experiments used to assign 15N, 13C, and 1H chemical shifts and interproton NOEs. This approach is tested experimentally on a 14 kDa SH2/phosphopeptide complex, and a global protein fold is obtained from a set of methyl-methyl, methyl-NH, and NH-NH distance restraints. We demonstrate that the inclusion of methyl-NH and methyl-methyl distance restraints greatly improves the precision and accuracy of structures relative to those generated with only NH-NH distance restraints. Finally, we examine the general applicability of this approach by determining the structures of several proteins with molecular masses of up to 40 kDa from simulated distance and dihedral angle restraint tables.
15N、13C、2H多维核磁共振波谱技术的发展，有助于对分子量超过30 kDa的蛋白质及蛋白质复合物的主链和侧链共振进行归属。这些方法的成功得益于高氘代蛋白质的制备；用氘取代与碳相连的质子，显著提高了化学位移归属中许多实验的灵敏度。不幸的是，均匀氘代也会从根本上减少可用于结构测定的质子间距离限制，降低所得结构的质量。在此，我们描述了一种方法，通过使用氘代、选择性甲基质子化的样品，提高从高氘代蛋白质确定的整体折叠结构的精度和准确性。这种标记模式保持了三共振核磁共振实验的效率，同时在可用于建立不同二级结构元件之间基于核Overhauser效应（NOE）接触的位置保留了足够数量的质子。我们评估了这种氘代方案如何影响用于归属15N、13C和1H化学位移以及质子间NOE的实验的灵敏度和分辨率。该方法在一个14 kDa的SH2/磷酸肽复合物上进行了实验测试，并从一组甲基-甲基、甲基-NH和NH-NH距离限制中获得了整体蛋白质折叠结构。我们证明，相对于仅使用NH-NH距离限制生成的结构，纳入甲基-NH和甲基-甲基距离限制大大提高了结构的精度和准确性。最后，我们通过从模拟距离和二面角限制表中确定几种分子量高达40 kDa的蛋白质的结构，检验了该方法的普遍适用性。
