基于甲基- trosy的13C弛豫色散核磁共振实验研究蛋白质中的化学交换

IF 1.3 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY
Vitali Tugarinov, James L. Baber, G. Marius Clore
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引用次数: 0

摘要

描述了一个基于甲基横向弛豫优化光谱(methyl- trosy)的多量子(MQ) 13C carr - purcell - meiboomm - gill (CPMG)弛豫色散核磁共振实验。该实验源自先前开发的MQ 13C - 1H CPMG方案(Korzhnev in J Am Chem Soc 126: 3964 - 73,2004),并辅以恒频重聚焦1H脉冲的CPMG序列,并与13C CPMG脉冲序列同步。最佳的1H“解耦”方案利用了重聚焦复合1H脉冲的XY-4相位循环,最大限度地减少了CPMG间隔期间存在的快速放松甲基MQ磁化量。对于中小型蛋白质,MQ 13C CPMG实验比单量子(SQ) 13C实验具有显著降低甲基相干的内在、无交换弛豫率的优势。对于高分子量蛋白质,MQ 13C CPMG实验消除了MQ 13C - 1H CPMG弛豫色散谱解释的复杂性,这些弛豫色散谱是由基态和激发态之间甲基1H化学位移差异对交换的贡献引起的。MQ 13C CPMG实验在两种蛋白质体系上进行了测试:(1)Fyn SH3结构域的三突变体在主要折叠态和激发态折叠中间体之间的化学位移时间尺度上缓慢相互转换;(2) 82 kda的苹果酸合成酶G (MSG),其中在单个Ile δ1甲基位置的化学交换发生的时间尺度要快得多。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins

A methyl-TROSY based 13C relaxation dispersion NMR experiment for studies of chemical exchange in proteins

A methyl Transverse Relaxation Optimized Spectroscopy (methyl-TROSY) based, multiple quantum (MQ) 13C Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiment is described. The experiment is derived from the previously developed MQ 13C–1H CPMG scheme (Korzhnev in J Am Chem Soc 126: 3964–73, 2004) supplemented with a CPMG train of refocusing 1H pulses applied with constant frequency and synchronized with the 13C CPMG pulse train. The optimal 1H ‘decoupling’ scheme that minimizes the amount of fast-relaxing methyl MQ magnetization present during CPMG intervals, makes use of an XY-4 phase cycling of the refocusing composite 1H pulses. For small-to-medium sized proteins, the MQ 13C CPMG experiment has the advantage over its single quantum (SQ) 13C counterpart of significantly reducing intrinsic, exchange-free relaxation rates of methyl coherences. For high molecular weight proteins, the MQ 13C CPMG experiment eliminates complications in the interpretation of MQ 13C–1H CPMG relaxation dispersion profiles arising from contributions to exchange from differences in methyl 1H chemical shifts between ground and excited states. The MQ 13C CPMG experiment is tested on two protein systems: (1) a triple mutant of the Fyn SH3 domain that interconverts slowly on the chemical shift time scale between the major folded state and an excited state folding intermediate; and (2) the 82-kDa enzyme Malate Synthase G (MSG), where chemical exchange at individual Ile δ1 methyl positions occurs on a much faster time-scale.

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来源期刊
Journal of Biomolecular NMR
Journal of Biomolecular NMR 生物-光谱学
CiteScore
6.00
自引率
3.70%
发文量
19
审稿时长
6-12 weeks
期刊介绍: The Journal of Biomolecular NMR provides a forum for publishing research on technical developments and innovative applications of nuclear magnetic resonance spectroscopy for the study of structure and dynamic properties of biopolymers in solution, liquid crystals, solids and mixed environments, e.g., attached to membranes. This may include: Three-dimensional structure determination of biological macromolecules (polypeptides/proteins, DNA, RNA, oligosaccharides) by NMR. New NMR techniques for studies of biological macromolecules. Novel approaches to computer-aided automated analysis of multidimensional NMR spectra. Computational methods for the structural interpretation of NMR data, including structure refinement. Comparisons of structures determined by NMR with those obtained by other methods, e.g. by diffraction techniques with protein single crystals. New techniques of sample preparation for NMR experiments (biosynthetic and chemical methods for isotope labeling, preparation of nutrients for biosynthetic isotope labeling, etc.). An NMR characterization of the products must be included.
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