Transverse relaxation optimized spectroscopy of NH2 groups in glutamine and asparagine side chains of proteins

IF 1.3 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Biomolecular NMR Pub Date : 2024-07-31 DOI:10.1007/s10858-024-00445-8

Vitali Tugarinov, Francesco Torricella, Jinfa Ying, G. Marius Clore

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引用次数: 0

Abstract

A transverse relaxation optimized spectroscopy (TROSY) approach is described for the optimal detection of NH₂ groups in asparagine and glutamine side chains of proteins. Specifically, we have developed NMR experiments for isolating the slow-relaxing ¹⁵N and ¹H components of NH₂ multiplets. Although even modest sensitivity gains in 2D NH₂-TROSY correlation maps compared to their decoupled NH₂–HSQC counterparts can be achieved only occasionally, substantial improvements in resolution of the NMR spectra are demonstrated for asparagine and glutamine NH₂ sites of a buried cavity mutant, L99A, of T4 lysozyme at 5 ºC. The NH₂-TROSY approach is applied to CPMG relaxation dispersion measurements at the side chain NH₂ positions of the L99A T4 lysozyme mutant — a model system for studies of the role of protein dynamics in ligand binding.

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蛋白质谷氨酰胺和天冬酰胺侧链中 NH2 基团的横向弛豫优化光谱。

本文介绍了一种横向弛豫优化光谱（TROSY）方法，用于优化蛋白质天冬酰胺和谷氨酰胺侧链中 NH2 基团的检测。具体来说，我们开发了 NMR 实验，用于分离 NH2 复合物中的慢弛豫 15N 和 1H 成分。虽然与解耦 NH2-HSQC 对应图相比，二维 NH2-TROSY 相关图的灵敏度偶尔也会略有提高，但对于 5 ºC 时 T4 溶菌酶的埋藏腔突变体 L99A 的天冬酰胺和谷氨酰胺 NH2 位点，核磁共振光谱的分辨率却有了大幅提高。NH2-TROSY 方法被应用于 L99A T4 溶菌酶突变体侧链 NH2 位点的 CPMG 松弛弥散测量，该突变体是研究配体结合中蛋白质动力学作用的模型系统。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Journal of Biomolecular NMR 生物-光谱学

CiteScore

6.00

自引率

3.70%

发文量

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.