通过甲基-TROSY核磁共振光谱探测高分子量蛋白质中的慢动力学：应用于一种723个残基的酶。

Probing slow dynamics in high molecular weight proteins by methyl-TROSY NMR spectroscopy: application to a 723-residue enzyme.

作者信息

Korzhnev Dmitry M, Kloiber Karin, Kanelis Voula, Tugarinov Vitali, Kay Lewis E

机构信息

Protein Engineering Network Centres of Excellence and the Departments of Medical Genetics, Biochemistry, and Chemistry, University of Toronto, Toronto, Ontario, Canada M5S 1A8.

出版信息

J Am Chem Soc. 2004 Mar 31;126(12):3964-73. doi: 10.1021/ja039587i.

DOI:10.1021/ja039587i

PMID:15038751

Abstract

A new CPMG-based multiple quantum relaxation dispersion experiment is presented for measuring millisecond dynamic processes at side-chain methyl positions in high molecular weight proteins. The experiment benefits from a methyl-TROSY effect in which cancellation of intramethyl dipole fields occurs, leading to methyl (13)C-(1)H correlation spectra of high sensitivity and resolution (Tugarinov, V.; Hwang, P. M.; Ollerenshaw, J. E.; Kay, L. E. J. Am. Chem. Soc. 2003, 125, 10420-10428). The utility of the methodology is illustrated with an application to a highly deuterated, methyl-protonated sample of malate synthase G, an 82 kDa enzyme consisting of a single polypeptide chain. A comparison of the sensitivity obtained using the present approach relative to existing HSQC-type (13)C single quantum dispersion experiments shows a gain of a factor of 5.4 on average, significantly increasing the range of applications for this methodology.

摘要

本文提出了一种基于CPMG的多量子弛豫色散实验，用于测量高分子量蛋白质侧链甲基位置的毫秒级动态过程。该实验受益于甲基-TROSY效应，其中甲基内偶极场相互抵消，从而产生高灵敏度和分辨率的甲基(13)C-(1)H相关谱（图加里诺夫，V.；黄，P. M.；奥勒伦肖，J. E.；凯，L. E. 《美国化学会志》2003年，125卷，10420 - 10428页）。通过将该方法应用于苹果酸合酶G的高度氘代、甲基质子化样品，说明了该方法的实用性。苹果酸合酶G是一种由单条多肽链组成的82 kDa酶。将使用本方法获得的灵敏度与现有的HSQC型(13)C单量子色散实验进行比较，结果表明平均增益因子为5.4，显著扩大了该方法的应用范围。