Tugarinov Vitali, Kay Lewis E
Department of Medical Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
J Am Chem Soc. 2006 Jun 7;128(22):7299-308. doi: 10.1021/ja060817d.
Experiments for quantifying the amplitudes of motion of methyl-containing side chains are presented that exploit the rich network of cross-correlated spin relaxation interactions between intra-methyl dipoles in highly deuterated, selectively 13CH2D- or 13CH3-labeled proteins. In particular, the experiments measure spin relaxation rates of degenerate 1H transitions in methyl groups that, for high-molecular-weight proteins, are very simply related to methyl three-fold symmetry axis order parameters. The methodology presented is applied to studies of dynamics in a pair of systems, including the 7.5-kDa protein L and the 82-kDa enzyme malate synthase G. Good agreement between 1H- and 2H-derived measures of side-chain order are obtained on highly deuterated proteins with correlation times exceeding approximately 10 ns (correlation coefficients greater than 0.95). Although 2H- and 13C-derived measures of side-chain dynamics are still preferred, the present work underscores the potential of using 1H relaxation for semiquantitative estimates of methyl side-chain flexibility, while the high level of consistency between the different spin probes of motion establishes the reliability of the dynamics parameters.
本文介绍了用于量化含甲基侧链运动幅度的实验，这些实验利用了高度氘代、选择性13CH2D或13CH3标记的蛋白质中甲基内偶极子之间丰富的交叉相关自旋弛豫相互作用网络。特别是，这些实验测量了甲基中简并1H跃迁的自旋弛豫率，对于高分子量蛋白质而言，这些弛豫率与甲基三重对称轴序参数非常简单地相关。所提出的方法应用于一对系统的动力学研究，包括7.5 kDa的蛋白质L和82 kDa的苹果酸合酶G。在相关时间超过约10 ns（相关系数大于0.95）的高度氘代蛋白质上，1H和2H衍生的侧链序测量结果之间取得了良好的一致性。虽然仍首选2H和13C衍生的侧链动力学测量方法，但本研究强调了利用1H弛豫对甲基侧链柔韧性进行半定量估计的潜力，同时不同运动自旋探针之间的高度一致性确立了动力学参数的可靠性。
