BACKBONE DYNAMICS OF PROTEINS AS STUDIED BY N-15 INVERSE DETECTED HETERONUCLEAR NMR-SPECTROSCOPY - APPLICATION TO STAPHYLOCOCCAL NUCLEASE
BACKBONE DYNAMICS OF PROTEINS AS STUDIED BY N-15 INVERSE DETECTED HETERONUCLEAR NMR-SPECTROSCOPY - APPLICATION TO STAPHYLOCOCCAL NUCLEASE
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DOI：
10.1021/bi00449a003
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发表时间：
1989-11-14
期刊：
影响因子：
2.9
通讯作者：
BAX, A
中科院分区：
文献类型：
--
作者：
KAY, LE;TORCHIA, DA;BAX, A
本文介绍了使用新的二维核磁共振（NMR）脉冲序列，以提供深入了解蛋白质动力学。所开发的序列允许测量大分子中单个核的弛豫特性，从而为局部蛋白质流动性的研究提供了一种强有力的实验方法。对于同位素标记的大分子，该序列能够测量异质结Overhauser效应（NOE）和自旋-晶格（T1）和自旋-自旋（T2）15 N或13 C弛豫时间，其灵敏度类似于许多homemandron 1H实验。由于T1值和异源NOE对高频运动（108-1012 s-1）敏感，而T2值也是慢得多的过程的函数，因此有可能探索在大时间尺度上发生的动态事件。我们已经应用这些技术来研究蛋白质葡萄球菌核酸酶（S。Nase）与胸苷3“-5”-二磷酸（pdTp）和Ca 2+复合，并用15 N均匀标记。T1，T2和NOE值，获得了超过100个指定的骨架酰胺氮的蛋白质。确定了表征~ 1H-~（15）N键快速运动程度的序参量（S）值。这些结果表明，这些快速的小振幅运动与次级结构之间没有相关性。纳塞相比之下，15 N线宽表明，在毫秒时间尺度上的二级结构与运动之间可能存在相关性。特别是，残基42和56之间的环区域在这个缓慢的时间尺度上似乎比蛋白质的其余部分更加灵活。
This paper describes the use of novel two-dimensional nuclear magnetic resonance (NMR) pulse sequence to provide insight into protein dynamics. The sequences developed permit the measurement of the relaxation properties of individual nuclei in macromolecules, thereby providing a powerful experimental approach to the study of local protein mobility. For isotopically labeled macromolecules, the sequences enable measurements of heteronuclear Overhauser effects (NOE) and spin-lattice (T1) and spin-spin (T2) 15N or 13C relaxation times with a sensitivity similar to those of many homonuclear 1H experiments. Because T1 values and heteronuclear NOEs are sensitive to high-frequency motions (108-1012 s-1) while T2 values are also a function of much slower processes, it is possible to explore dynamic events occurring over a large time scale. We have applied these techniques to investigate the backbone dynamics of the protein staphylococcal nuclease (S. Nase) complexed with thymidine 3''-5''-bisphosphate (pdTp) and Ca2+ and labeled uniformly with 15N. T1, T2, and NOE values were obtained for over 100 assigned backbone amide nitrogens in the protein. Values of the order parameter (S), characterizing the extent of rapid 1H-15N bond motions, have been determined. These results suggest that there is no correlation between these rapid small amplitude motions and secondary structure for S. Nase. In contrast, 15N line widths suggest a possible correlation between secondary structure ans motions on the millisecond time scale. In particular, the loop region between residues 42 and 56 appears to be considerably more flexible on this slow time scale than the rest of the protein.