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Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques

机译：具有批量和单分子技术的整合使用蛋白质动力学

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摘要

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.
机译：蛋白质动态地样本多构象，构象动态使蛋白质功能能够。大多数生物物理测量都是基于合奏的，观察到对集合的所有成员进行平均。虽然可达到，但可观察到的组成符合状态的分解可以计算得昂贵和含糊不清。在这里，我们表明，掺入单分子荧光共振能量转移（SMFRet）数据解决了模糊性并提供更精确和准确的蛋白质集合结构。使用K63连接的二聚蛋白，我们表征了模型系统的动态域布置，使用化学交联与质谱（CXMS），小角度X射线散射（SAX）和SMFRet技术耦合。 CXMS允许蛋白质构象状态建模，这些状态是晶体结构的替代品。萨克斯提供集合平均的低分辨率形状信息。重要的是，SMFRet提供了特定于特定的群体，并且FRET距离通过改进CXMS和SAXS约束来验证所获得的集合结构。批量和单分子技术的一致性使用能够更好地了解蛋白质动态，并在结构生物学中广泛实现。

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  • 来源
    《Biochemistry》 |2018年第3期|共9页
  • 作者

    Zhu Liu; Zhou Gong; Yong Cao; Yue-He Ding; Meng-Qiu Dong; Yun-Bi Lu; Wei-Ping Zhang; Chun Tang;

  • 作者单位

    CAS Key Laboratory of Magnetic Resonance in Biological Systems State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics and National Center for Magnetic Resonance at Wuhan Wuhan Institute of Physics and Mathematics of the Chinese Academy;

    CAS Key Laboratory of Magnetic Resonance in Biological Systems State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics and National Center for Magnetic Resonance at Wuhan Wuhan Institute of Physics and Mathematics of the Chinese Academy;

    National Institute of Biological Sciences Beijing 102206 China;

    National Institute of Biological Sciences Beijing 102206 China;

    National Institute of Biological Sciences Beijing 102206 China;

    Department of Pharmacology Institute of Neuroscience Key Laboratory of Medical Neurobiology of Ministry of Health of China and Zhejiang Province Key Laboratory of Mental Disorder’s Management Zhejiang University School of Medicine Hangzhou Zhejiang;

    Department of Pharmacology Institute of Neuroscience Key Laboratory of Medical Neurobiology of Ministry of Health of China and Zhejiang Province Key Laboratory of Mental Disorder’s Management Zhejiang University School of Medicine Hangzhou Zhejiang;

    CAS Key Laboratory of Magnetic Resonance in Biological Systems State Key Laboratory of Magnetic Resonance and Atomic Molecular Physics and National Center for Magnetic Resonance at Wuhan Wuhan Institute of Physics and Mathematics of the Chinese Academy;

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  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;
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