类细胞环境下蛋白质结构与功能的NMR研究

徐国华; 李从刚; 刘买利

doi:10.7536/PC161225

化学进展 ›› 2017, Vol. 29 ›› Issue (1) :75-82 DOI: 10.7536/PC161225

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类细胞环境下蛋白质结构与功能的NMR研究

徐国华, 李从刚*, 刘买利*

作者信息 +

NMR Study of Protein Structure and Function in Cell-Like Environment

Guohua Xu, Conggang Li*, Maili Liu*

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文章历史 +

摘要

细胞环境是大多数蛋白质行使其功能的天然环境。在细胞环境中，大分子拥挤、限域、弱相互作用等普遍存在，已经有越来越多的研究表明这种复杂细胞环境会影响蛋白质的结构与功能。因此，在越接近天然的环境中研究蛋白质，越有可能揭示蛋白质功能的本质。蛋白质结构与功能的原位研究已是目前蛋白质科学中的前沿领域。核磁共振波谱技术是研究复杂环境中蛋白质结构与功能最具潜力的手段，它能提供原子分辨的蛋白质结构，动态与相互作用信息。本文总结了我们近几年利用核磁共振技术研究大分子拥挤、限域、以及原核E.coli细胞、非洲爪蟾卵母细胞中蛋白质结构与功能等方面取得的重要进展。

Abstract

Most proteins function in the cell where macromolecular crowding, confinement and quinary interaction are ubiquitous. More and more researches suggest that the complex cellular environment affects protein's structure and function. Therefore, for protein studies, the closer to native cellular environment, the more likely molecular mechanisms of proteins function could be revealed accurately. In-cell study of protein structure and function has been a frontier topic in protein science. Nuclear magnetic resonance (NMR) spectroscopy is the most promising technique for protein structural and functional assay at atomic level in complex environments. Here, we summarize our recent progress in protein structural and functional studies in macromolecular crowding, confinement, prokaryotic and eukaryotic cells by NMR spectroscopy. Two model proteins, an intrinsic disordered protein α-synuclein and a multi-domain protein calmodulin, are employed to show how macromolecular crowding and confinement affect protein structure and function, respectively. Then in-cell NMR methods, including labeling strategy, cytoplasmic viscosity measurement, quinary interaction quantification are developed to obtain high-quality NMR spectra for facilitating protein structural and functional studies in living cells. Ca²⁺-induced calmodulin conformational transitions and GB1 protein structural determination in living Xenopus laevis oocytes, are shown here as typical applications of in-cell NMR. Finally, the conclusion and perspective of environmental effects on protein structure and function are presented.

Contents
1 Introduction
2 The effect of macromolecular crowding on protein structure and function
3 The effect of confinement on protein structure and function
4 In vivo NMR study of protein structure and function
4.1 Labeling strategies for in-cell protein NMR study
4.2 The determination of cytoplasmic viscosity and weak protein interactions in living cells
4.3 The observation of Ca²⁺-induced calmodulin conformational transitions in intact Xenopus laevis oocytes
4.4 Direct determination of protein structure in living Xenopus laevis oocytes
5 Conclusion

Graphical abstract

关键词

核磁共振波谱 / 大分子拥挤 / 限域 / 五级相互作用 / 蛋白质的结构和功能

Key words

nuclear magnetic resonance (NMR) spectroscopy / macromolecular crowding / confinement / quinary interaction / protein structure and function

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徐国华, 李从刚, 刘买利. 类细胞环境下蛋白质结构与功能的NMR研究[J]. 化学进展, 2017, 29(1): 75-82 DOI:10.7536/PC161225

Guohua Xu, Conggang Li, Maili Liu. NMR Study of Protein Structure and Function in Cell-Like Environment[J]. Progress in Chemistry, 2017, 29(1): 75-82 DOI:10.7536/PC161225

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