DYNAMICS OF METHYL-GROUPS IN PROTEINS AS STUDIED BY PROTON-DETECTED C-13 NMR-SPECTROSCOPY - APPLICATION TO THE LEUCINE RESIDUES OF STAPHYLOCOCCAL NUCLEASE
DYNAMICS OF METHYL-GROUPS IN PROTEINS AS STUDIED BY PROTON-DETECTED C-13 NMR-SPECTROSCOPY - APPLICATION TO THE LEUCINE RESIDUES OF STAPHYLOCOCCAL NUCLEASE
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DOI：
10.1021/bi00138a003
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发表时间：
1992-06-16
期刊：
影响因子：
2.9
通讯作者：
TORCHIA, DA
中科院分区：
文献类型：
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作者：
NICHOLSON, LK;KAY, LE;TORCHIA, DA
本文介绍了最近发展的核磁共振脉冲序列的应用，以获得有关蛋白质中富含同位素的疏水侧链的内部动力学信息。脉冲序列提供的二维光谱使人们能够准确地测量蛋白质中丰富的甲基碳的核Overhauser效应(NOE)和纵向(T1)和横向(T2)弛豫时间。本文用这些技术研究了葡萄球菌核酸酶(SNase)的11个亮氨酸侧链的内部动力学，SNase是一种M(R)=16.8K的小酶，在没有和存在配体3‘，5’-二磷酸(PdTp)和钙离子的情况下。我们报道了[5，5‘-C-13(2)]亮氨酸的合成，含有标记亮氨酸的SNase的制备，连接和未连接蛋白质中亮氨酸甲基碳和质子的顺序归属，以及SNase亮氨酸甲基碳的C-13T1、T2和NOE值的测定。用Lipari和Szabo的形式对弛豫参数的分析表明，亮氨酸甲基碳的内部运动由有效关联时间tau(F)(5-80ps)和tau(S)(
This paper describes the application of recently developed nuclear magnetic resonance (NMR) pulse sequences to obtain information about the internal dynamics of isotopically enriched hydrophobic side chains in proteins. The two-dimensional spectra provided by the pulse sequences enable one to make accurate measurements of nuclear Overhauser effects (NOE) and longitudinal (T1) and transverse (T2) relaxation times of enriched methyl carbons in proteins. Herein, these techniques are used to investigate the internal dynamics of the 11 leucine side chains of staphylococcal nuclease (SNase), a small enzyme having M(r) = 16.8K, in the absence and presence of ligands thymidine 3',5'-bisphosphate (pdTp) and Ca2+. We report the synthesis of [5,5'-C-13(2)]leucine, the preparation of SNase containing the labeled leucine, the sequential assignment of the leucine methyl carbons and protons in the liganded and unliganded proteins, and the measurement of the C-13 T1, T2, and NOE values for the SNase leucine methyl carbons. Analysis of the relaxation parameters using the formalism of Lipari and Szabo shows that the internal motions of the leucine methyl carbons are characterized by effective correlation times tau(f) (5-80 ps) and tau(s) (