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首页> 外文学位 >Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule Experiments and Simulations to Bridge Timescales.
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Conformation and Dynamics of the Troponin I C-Terminal Domain: Combining Single-Molecule Experiments and Simulations to Bridge Timescales.

机译：肌钙蛋白I C末端结构域的构象和动力学：结合单分子实验和模拟来桥接时间尺度。

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摘要

Troponin is a heterotrimeric complex that acts as a molecular switch on the thin filament of striated muscle, linking myosin-based contractile function with calcium signaling. The activation of the thin filament can be broadly described in three stages: blocked, closed, and open, each characterized by a tropomyosin position that permits or restricts myosin access. Troponin, particularly the troponin I subunit, undergoes large conformational changes to move tropomyosin through these activation states and permit the cyclical contraction and relaxation cycle of the heartbeat. However, many details of troponin's function remain uncertain.;The C-terminus of troponin I (TnI-C) is a roughly fifty-residue long domain believed to undergo a disorder-order transition during muscle relaxation (moving from a free, unbound conformation in the open state to a folded, bound conformation in the blocked state). It is believed that this transition progresses via a fly-casting mechanism, in which the disorder in TnI-C's open state allows it to locate its binding site on the thin filament quickly. TnI-C binding the thin filament may be the event that begins the sequence of conformational changes leading to the shifting of tropomyosin and inhibition of the thin filament, making this transition highly important in understanding muscle function at a molecular level. This transition is also believed to be dysregulated in hypertrophic cardiomyopathy, a genetic cardiac disease whose underlying molecular mechanism is poorly understood. Therefore, understanding the TnI-C fly-casting mechanism is important from both basic science and clinical perspectives.;This dissertation details our efforts to study the open state of TnI-C. The disordered state of a protein undergoing coupled binding and folding can define the kinetics and binding mechanism of the interaction. Here, we use a combined approach of single-molecule fluorescence and simulations to elucidate the conformation and dynamics of TnI-C in its disordered state, drawing upon a history of intrinsically disordered protein research that has informed many relevant techniques and approaches. We detail new methods for comparing simulations and experiments differing in timescales by orders of magnitude, and develop new analyses to evaluate intrinsically disordered regions, which have varying degrees of disorder throughout their sequences.;Finally, we present a picture of TnI-C in the open conformation of the solution-state troponin complex. TnI-C is a highly dynamic, extended domain, well poised for thin filament interaction. While disordered, TnI-C frequently samples three conformations, which are qualitatively similar to previous publications of contradictory TnI-C "structures". We show that even with these three clusters of conformations, TnI-C is globally rather homogeneous in its conformation, and that short molecular dynamics trajectories can probe its underlying dynamics. These analyses uncover extensive negatively correlated motion in the domain, driven in part by charge distribution, that is responsible for maintaining TnI-C in its extended conformation. This reliance on charge for TnI-C's conformation suggests a potential for alterations through targeted mutations or small molecules, both of which could be useful in the laboratory or as pharmaceutical targets for disease. We conclude by presenting potential directions for future research into this intrinsically disordered region, which could serve as a model protein for understanding coupled binding and folding as well as serve a medical purpose.
机译：肌钙蛋白是异三聚体复合物，可作为横纹肌细丝上的分子开关，将基于肌球蛋白的收缩功能与钙信号传导联系起来。细丝的激活可以大致分为三个阶段：封闭，闭合和开放，每个阶段的特征是原肌球蛋白位置允许或限制肌球蛋白的进入。肌钙蛋白，尤其是肌钙蛋白I亚基，经历了较大的构象变化，以使肌钙蛋白移动通过这些激活状态，并允许心跳的周期性收缩和松弛周期。然而，肌钙蛋白功能的许多细节仍不确定。肌钙蛋白I（TnI-C）的C端是大约50个残基的长域，据信在肌肉松弛过程中会经历无序秩序的转变（从自由的，未结合的构象转变而来）在开放状态下折叠为约束状态（在封闭状态下）。据信，这种转变是通过飞铸机制进行的，其中TnI-C开放状态的紊乱使它能够迅速将其结合位点定位在细丝上。 TnI-C结合细丝可能是开始构象变化的事件，导致原肌球蛋白转移并抑制了细丝，从而使这种转变在分子水平上理解肌肉功能非常重要。人们还认为，这种转变在肥厚型心肌病中是失调的，肥厚型心肌病是一种遗传性心脏病，其潜在的分子机制尚不清楚。因此，从基础科学和临床角度了解TnI-C的飞铸机理都具有重要意义。经历偶联结合和折叠的蛋白质的无序状态可以定义相互作用的动力学和结合机理。在这里，我们使用结合了单分子荧光和模拟的方法，阐明了处于无序状态的TnI-C的构象和动力学，并借鉴了许多相关技术和方法所固有的无序蛋白质研究的历史。我们详细介绍了比较时间尺度不同数量级的模拟和实验的新方法，并开发了新的分析方法来评估固有无序区域，这些区域在整个序列中具有不同程度的无序性;最后，我们给出了TnI-C的图片溶液状态肌钙蛋白复合物的开放构象。 TnI-C是一个高度动态的扩展域，非常适合细丝相互作用。尽管混乱，TnI-C经常采样三个构象，它们在质量上与先前的相互矛盾的TnI-C“结构”出版物相似。我们表明，即使具有这三个构象簇，TnI-C的构象在全局上也是相当均一的，并且短分子动力学轨迹可以探测其潜在的动力学。这些分析揭示了部分受电荷分布驱动的域中广泛的负相关运动，该运动负责使TnI-C保持其扩展构象。 TnI-C构象对电荷的依赖表明有可能通过靶向突变或小分子发生改变，这两种突变都可用于实验室或作为疾病的药物靶标。最后，我们提出了对该内在无序区域进行未来研究的潜在方向，该方向可作为模型蛋白，用于理解偶联结合和折叠以及医学目的。

著录项

  • 作者

    Metskas, Lauren Ann.;

  • 作者单位

    Yale University.;

  • 授予单位 Yale University.;
  • 学科 Biophysics.
  • 学位 Ph.D.
  • 年度 2016
  • 页码 227 p.
  • 总页数 227
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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