Measurement of slow (μs-ms) time scale dynamics in protein side chains by 15N relaxation dispersion NMR spectroscopy:: Application to Asn and Gln residues in a cavity mutant of T4 lysozyme
Measurement of slow (μs-ms) time scale dynamics in protein side chains by 15N relaxation dispersion NMR spectroscopy:: Application to Asn and Gln residues in a cavity mutant of T4 lysozyme
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DOI：
10.1021/ja003447g
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发表时间：
2001-02-07
影响因子：
15
通讯作者：
Kay, LE
中科院分区：
文献类型：
--
作者：
Mulder, FAA;Skrynnikov, NR;Kay, LE
提出了一种新的 NMR 实验，用于测量蛋白质中 Asn 和 Gin 侧链的 mus-ms 时间尺度动态。交换对侧链残基 N-15 线宽的贡献是通过弛豫分散实验确定的，其中有效氮横向弛豫率是作为恒定时间、可变间距 CPMG 间隔中重聚焦脉冲数量的函数来测量的。标量耦合和偶极-偶极互相关产生的磁化强度过去限制了多自旋系统中交换的研究，但并不影响从本实验获得的NH2基团弛豫曲线中提取准确的交换参数。通过对 T4 溶菌酶 L99A 的 Leu --> Ala 空腔突变体的应用，证明了该方法的实用性。 II 显示蛋白质 C 端结构域中 Asn 和 Gin 残基的许多侧链酰胺基团受到化学交换过程的影响，这对于促进疏水性配体与空腔的快速结合可能很重要。
A new NMR experiment is presented for the measurement of mus-ms time scale dynamics of Asn and Gin side chains in proteins. Exchange contributions to the N-15 line widths of side chain residues are determined via a relaxation dispersion experiment in which the effective nitrogen transverse relaxation rate is measured as a function of the number of refocusing pulses in constant-time, variable spacing CPMG intervals. The evolution of magnetization from scalar couplings and dipole-dipole cross-correlations, which has limited studies of exchange in multi-spin systems in the past, does not affect the extraction of accurate exchange parameters from relaxation profiles of NH2 groups obtained in the present experiment. The utility of the method is demonstrated with an application to a Leu --> Ala cavity mutant of T4 lysozyme, L99A. II is shown that many of the side chain amide groups of Asn and Gin residues in the C-terminal domain of the protein are affected by a chemical exchange process which may be important in facilitating the rapid binding of hydrophobic ligands to the cavity.