Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein.
Quantitative comparison between sub-millisecond time resolution single-molecule FRET measurements and 10-second molecular simulations of a biosensor protein.
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DOI：
10.1371/journal.pcbi.1008293
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发表时间：
2020-11
影响因子：
4.3
通讯作者：
Sanbonmatsu KY
中科院分区：
文献类型：
--
作者：
Girodat D;Pati AK;Terry DS;Blanchard SC;Sanbonmatsu KY
分子动力学(MD)模拟试图以实验上相关的时间分辨率，如单分子荧光测量提供的时间分辨率，提供对构象动态生物系统的原子水平的洞察。然而，分子动力学模拟的时间尺度和单分子测量的时间分辨率的限制对获得密切定量比较所需的重叠时间区域的努力提出了挑战。实现这种重叠有可能提供新的理论、假设和解释，从而使理想化的实验设计能够最大限度地检测所需的反应坐标。在这里，我们报告了与氨基酸结合蛋白Liv-BPSS的体外单分子Förster(荧光)共振能量转移(SmFRET)测量重叠的时间尺度的MD模拟，分辨率为亚毫秒。采用了计算高效的全原子结构模拟，并与显式溶剂模拟进行了对比，对多个周期的LiV-BPSS翻盖状构象变化在秒的时间尺度上进行了采样，检查了这些事件与smFRET观察到的事件之间的关系。MD模拟与smFRET的测量结果一致，并提供了关于荧光团在Liv-BPSS上附着位置的局部动力学以及荧光团运动与Liv-BPSS结构域之间的大尺度构象变化之间的关联的有价值的信息。我们进一步利用MD模拟来解释SMFRET数据，包括Förster半径(R0)和荧光团取向因子(κ2)的确定。我们描述的方法可以很容易地扩展到不同的生化系统，允许解释任何与蛋白质或核糖核蛋白复合体结合的FRET系统，包括那些具有更多构象过程的系统，以及那些实现多颜色smFRET的系统。Förster(荧光)共振能量转移(FRET)被生物物理学家广泛用作分子尺度的标尺，对生物功能所需的复杂形成和构象重排等瞬变过程提供基本的结构和动力学方面的见解。FRET技术需要识别信息丰富的荧光团标记位置，以确定距离以告知感兴趣的反应坐标，并考虑可能模糊定量解释的噪声源。在这里，我们描述了一种利用计算高效的基于全原子结构的分子动力学模拟的进步的方法，其中通过FRET观察到的结构动力学可以在相称的时间尺度上被完整的原子细节解释。我们使用一个模型FRET系统，将氨基酸结合蛋白Liv-BPSS连接到自修复的有机荧光团，展示了这种方法的潜力。LiV-BPSS在开放构象和闭合构象之间表现出大规模的亚毫秒翻盖状构象变化，分别与配体解离和结合有关。我们的发现基于smFRET观察到的动力学的分子基础，以及优化荧光团标记位置、荧光团附着方式和荧光团组成的策略。
Molecular Dynamics (MD) simulations seek to provide atomic-level insights into conformationally dynamic biological systems at experimentally relevant time resolutions, such as those afforded by single-molecule fluorescence measurements. However, limitations in the time scales of MD simulations and the time resolution of single-molecule measurements have challenged efforts to obtain overlapping temporal regimes required for close quantitative comparisons. Achieving such overlap has the potential to provide novel theories, hypotheses, and interpretations that can inform idealized experimental designs that maximize the detection of the desired reaction coordinate. Here, we report MD simulations at time scales overlapping with in vitro single-molecule Förster (fluorescence) resonance energy transfer (smFRET) measurements of the amino acid binding protein LIV-BPSS at sub-millisecond resolution. Computationally efficient all-atom structure-based simulations, calibrated against explicit solvent simulations, were employed for sampling multiple cycles of LIV-BPSS clamshell-like conformational changes on the time scale of seconds, examining the relationship between these events and those observed by smFRET. The MD simulations agree with the smFRET measurements and provide valuable information on local dynamics of fluorophores at their sites of attachment on LIV-BPSS and the correlations between fluorophore motions and large-scale conformational changes between LIV-BPSS domains. We further utilize the MD simulations to inform the interpretation of smFRET data, including Förster radius (R0) and fluorophore orientation factor (κ2) determinations. The approach we describe can be readily extended to distinct biochemical systems, allowing for the interpretation of any FRET system conjugated to protein or ribonucleoprotein complexes, including those with more conformational processes, as well as those implementing multi-color smFRET. Förster (fluorescence) resonance energy transfer (FRET) has been used extensively by biophysicists as a molecular-scale ruler that yields fundamental structural and kinetic insights into transient processes including complex formation and conformational rearrangements required for biological function. FRET techniques require the identification of informative fluorophore labeling sites, spaced at defined distances to inform on a reaction coordinate of interest and consideration of noise sources that have the potential to obscure quantitative interpretations. Here, we describe an approach to leverage advancements in computationally efficient all-atom structure-based molecular dynamics simulations in which structural dynamics observed via FRET can be interpreted in full atomistic detail on commensurate time scales. We demonstrate the potential of this approach using a model FRET system, the amino acid binding protein LIV-BPSS conjugated to self-healing organic fluorophores. LIV-BPSS exhibits large scale, sub-millisecond clamshell-like conformational changes between open and closed conformations associated with ligand unbinding and binding, respectively. Our findings inform on the molecular basis of the dynamics observed by smFRET and on strategies to optimize fluorophore labeling sites, the manner of fluorophore attachment, and fluorophore composition.
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DOI：
10.1039/c8cp04112a
发表时间：
2018-10-31
期刊：
Physical chemistry chemical physics : PCCP
影响因子：
--
作者：
Gahlon HL ;Walker AR ;Cisneros GA ;Lamers MH ;Rueda DS
通讯作者：
Rueda DS
影响因子：
5.5
作者：
Hess, Berk;Kutzner, Carsten;Lindahl, Erik
通讯作者：
Lindahl, Erik
影响因子：
64.8
作者：
Bao H;Das D;Courtney NA;Jiang Y;Briguglio JS;Lou X;Roston D;Cui Q;Chanda B;Chapman ER
通讯作者：
Chapman ER
影响因子：
5.5
作者：
Bowerman, Samuel;Rana, Ambar S. J. B.;Wereszczynski, Jeff
通讯作者：
Wereszczynski, Jeff
影响因子：
--
作者：
BRYNGELSON, JD;WOLYNES, PG
通讯作者：
WOLYNES, PG