物理化学学报

所属专题: 固体核磁共振 化学科普(2019)

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基于固体核磁共振方法的蛋白质组装体三维结构解析

邓静1,2, 马涛1,2, 常自伟1, 赵伟静1, 杨俊1   

  1. 1 中国科学院武汉物理与数学研究所, 波谱与原子分子物理国家重点实验室, 武汉磁共振中心, 武汉 430071;
    2 中国科学院大学, 北京 100049
  • 收稿日期:2019-05-02 修回日期:2019-06-10 录用日期:2019-06-17 发布日期:2019-06-24
  • 通讯作者: 杨俊 E-mail:yangjun@wipm.ac.cn
  • 基金资助:
    国家自然科学基金(21425523,31770798,31600613,21603269)资助项目

Determination of Three-Dimensional Structures of Protein Assemblies via Solid-State NMR

DENG Jing1,2, MA Tao1,2, CHANG Ziwei1, ZHAO Weijing1, YANG Jun1   

  1. 1 Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, National Center for Magnetic Resonance in Wuhan, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China;
    2 University of Chinese Academy of Sciences, Beijing 100049, P. R. China
  • Received:2019-05-02 Revised:2019-06-10 Accepted:2019-06-17 Published:2019-06-24
  • Contact: YANG Jun E-mail:yangjun@wipm.ac.cn
  • Supported by:
    The project was supported by the National Natural Science Foundation of China (21425523, 31770798, 31600613, 21603269).

摘要: 蛋白质组装体广泛存在于生物体内,具有相关生物学功能或与人类的重要疾病密切相关。蛋白质组装体分子量大,通常难以溶解和结晶,限制了常用的结构研究手段如X射线晶体学和液体NMR等在其高分辨三维结构解析中的应用。固体核磁共振技术(ssNMR)在难溶、非结晶样品的三维结构解析中具有独特的优势,尤其随着固体NMR硬件包括高场磁体和高性能的探头、固体NMR多维脉冲实验技术和样品制备技术特别是同位素标记技术的快速发展,固体NMR已经成为了蛋白组装体三维结构解析的重要手段。在样品制备方法方面,强调了样品制备条件的优化对得到构象均一样品的重要性,以及丰富的同位素标记方法的使用对固体NMR谱图分辨率提高的重要作用。同时多种脉冲序列如质子驱动自旋扩散技术(PDSD),偶极辅助旋转共振技术(DARR),质子辅助重偶技术(PAR)或转移回波双共振技术(TEDOR)等的建立和发展为结构约束条件收集提供了基本的技术方法。此外,固体NMR与其它实验技术如扫描透射电镜(STEM),冷冻电镜(Cryo-EM)等和理论模拟方法的联用能显著地提高固体NMR的能力,从而能解析分子量更大、结构更复杂的蛋白质组装体的三维结构。本文以Aβ纤维和T3SS针状体的三维结构解析为例介绍固体NMR在蛋白质组装体结构研究的最新实验方法,重点介绍最新的距离约束条件获取的实验方法进展,以及固体NMR与其它实验和理论模拟研究手段的联用在蛋白质组装体结构解析上的最新进展,期望有助于读者对固体NMR技术在蛋白质组装体的三维结构解析方面的研究进展有所了解。

关键词: 固体核磁共振, 蛋白质组装体, 高分辨结构, T3SS针状体, Aβ淀粉样纤维

Abstract: Biological assemblies with specific function or pathogenicity are widespread within organisms; however, their insolubility, amorphous properties, and large size are the major obstacles for structure determination via solution NMR spectroscopy and X-ray crystallography. In contrast, solid-state NMR (ssNMR) spectroscopy is not limited by the solubility or crystallinity of the sample and is a potent method to determine the structure of protein assemblies at atomic resolution. High magnetic field, fast magic-angle spinning (MAS), isotope labeling schemes, and improved methodology in ssNMR have enabled resonance assignment and restraints in structure determination among protein assemblies.
This review first discusses methods of obtaining structural restraints by ssNMR. Optimization of sample preparation is an effective approach to increase homogeneity in the conformation, thus also improving the resolution of ssNMR spectra. Furthermore, the resolution of 13C spectra can be further improved by using 13C sparse labeling strategies with selective labeling of carbon sources during protein expression. Structure characterization by ssNMR is based on structural restraints via multidimensional experiments correlating resonance between 13C and 15N. Protein secondary structure can be ascertained through chemical shifts involving 13Cα, 13Cβ, 13C', and 15N. The backbone torsion angle can be predicted using TALOS+ based on these chemical shifts. Site-specific structural restraints are accessible from 2D experiments such as 13C-13C, e.g., proton-driven spin diffusion (PDSD), dipolar-assisted rotational resonance (DARR), proton-assisted recoupling (PAR) and 13C-15N, e.g., transferred-echo double-resonance (TEDOR), rotational-echo double-resonance (REDOR). An additional issue is to distinguish inter-molecular and intra-molecular restraints. Preparations of mixed labeled samples (e.g., 50% 13C uniformly labeled subunits and 50% uniformly 15N labeled subunits) have yielded abundant structural restraints from ssNMR data, facilitating high-resolution structural analysis. Further, hybrid approaches based on ssNMR are discussed. Electron microscopy (EM) is a suitable method to investigate structural features including the diameter of the protein assemblies, which is "invisible" through ssNMR analysis. Scanning transmission electron microscopy (STEM) can help determine the mass-per-length parameters (MPL) of unbranched fibrils, thus confirming the number of subunits in a layer of fibrils. Cryo-EM is a powerful technique to describe the molecular envelope of protein assemblies. Cryo-EM potentially yields the density map and long-range symmetry parameters, while ssNMR provides atomic-level structural details; hence, Cryo-EM and ssNMR are highly complementary methods. X-ray diffraction can help determine the distance (4.5-4.7 Å, 1 Å=0.1 nm) along the fibril axis between adjacent polypeptide chains in β-strand conformation, generally referred to as the "cross-β" structure. Rosetta has simulated the protein structure in accordance with structural data obtained from protein data bank (PDB) with the same peptide sequence. On combining ssNMR with those methods, more abundant structural information may be obtained, thus shortening the structural calculation cycle. Finally, a detailed description of the ssNMR structural data on amyloid-β (Aβ) fibrils and T3SS needles are provided as examples. Various structural characteristics of Aβ40/Aβ42 were reported by several groups, including the trimeric or dimeric conformations, parallel or antiparallel, in-register or out-of-register arrangements of the β-strands, demonstrating the structural polymorphism of Aβ40/Aβ42. Atomic-resolution structures of T3SS needles were analyzed on the basis of high-resolution spectra, using 13C sparse-labeled and ssNMR-Cryo-EM-Rosetta hybrid approaches, indicating that hybrid approaches based on ssNMR are a powerful tool to determine the high-resolution structure of protein assemblies.

Key words: Solid-state NMR, Protein assembly, High resolution structure, Type III secretion system needle, β-amyloid fibril

MSC2000: 

  • O641