Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme.
Slow internal dynamics in proteins: application of NMR relaxation dispersion spectroscopy to methyl groups in a cavity mutant of T4 lysozyme.
复制标题
蛋白质内部动力学缓慢：NMR 弛豫色散光谱在 T4 溶菌酶空腔突变体中甲基的应用。
DOI：
10.1021/ja0119806
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发表时间：
2002
影响因子：
15
通讯作者：
Kay,LewisE
中科院分区：
文献类型：
--
作者：
Mulder,FransAA;Hon,Bin;Mittermaier,Anthony;Dahlquist,FrederickW;Kay,LewisE
最近发展的碳横向弛豫弥散实验(Skrynnikov，N.R.；et al.J.Am化学。Soc.2001,123,4556，−4566)，以甲基为动力学探针，研究了T4溶菌酶(L99A)空穴突变体的毫秒到微秒时间尺度的运动。表达的蛋白质是因。以13CH3-丙酮酸为唯一碳源的结肠癌细胞在总共80个Valγ、Leuδ、Ileγ2、Alaβ和Metε甲基位置上有高水平的13C浓缩，几乎没有外来掺入。有72个甲基的数据可供分析。测量了其中许多残基的大幅度色散分布，并很好地符合两态交换模型。通过对每个探针的松弛色散曲线进行拟合，得到的状态间转化率和态的粒子数非常均匀，几乎所有蛋白质中甲基的数据都可以集体拟合到单个合作构象转变。本研究证明了甲基弛豫色散测量对于研究蛋白质在大量侧链位置的毫秒时间尺度运动的普遍适用性。描述了与实验相关的潜在伪影，并提出了将其影响降至最低的方法。由于甲基的良好的核磁共振光谱特性，这些实验应该特别适合于在高分子量系统中的探测动力学。
Recently developed carbon transverse relaxation dispersion experiments (Skrynnikov, N. R.; et al.J. Am. Chem. Soc.2001,123, 4556−4566) were applied to the study of millisecond to microsecond time scale motions in a cavity mutant of T4 lysozyme (L99A) using methyl groups as probes of dynamics. Protein expressed inE. colicells with13CH3-pyruvate as the sole carbon source contained high levels of13C enrichment at a total of 80 Valγ, Leuδ, Ileγ2, Alaβ, and Metε methyl positions with little extraneous incorporation. Data for 72 methyl groups were available for analysis. Dispersion profiles with large amplitudes were measured for many of these residues and were well fit to a two-state exchange model. The interconversion rates and populations of the states, obtained from fitting relaxation dispersion profiles of each individual probe, were remarkably homogeneous and data for nearly all methyl groups in the protein could be collectively fit to a single cooperative conformational transition. The present study demonstrates the general applicability of methyl relaxation dispersion measurements for the investigation of millisecond time scale protein motions at a large number of side-chain positions. Potential artifacts associated with the experiments are described and methods to minimize their effects presented. These experiments should be particularly well suited for probing dynamics in high molecular weight systems due to the favorable NMR spectroscopic properties of methyl groups.