NSF-BSF: Synergistic Multiscale Modeling and Single-Molecule Fluorescence Studies of the Dynamics and Function of AAA+ Protein Disaggregation Machines

分解有毒蛋白质聚集体是确保细胞在应激条件下存活的重要质量控制机制。这一作用是由环状AAA+(与多种细胞活动相关的ATPase)生物纳米机器执行的，例如CLP/Hsp100(细菌中的ClpB或酵母中的Hsp104)，它们施加机械力从聚集体中提取蛋白质分子，并通过狭窄的毛孔转移它们，以帮助其复性过程。在微观水平上了解纳米机器的构象动力学与底物蛋白(SP)穿线和分解机制之间的耦合，将有助于阐明关键细胞过程的基本方面。该项目将协同结合在美国辛辛那提大学斯坦教授的实验室中进行的混合多尺度计算机模拟，以及在以色列魏兹曼研究所哈兰教授的实验室中进行的单分子荧光共振能量转移(SmFRET)实验。与该项目生物物理研究相结合的教育和指导活动的核心是增加少数群体对计算科学的参与。这些活动将包括在中央州立大学(CSU)的外联活动，这是一个历史上的黑人学院和大学，以及在辛辛那提大学为代表不足的少数族裔提供研究经验机会。此外，还将在辛辛那提博物馆中心为夏令营学生提供科学培训计划。该项目中的美以交流项目将为学生和博士后提供跨学科的经验和国际视野。这个项目将解决蛋白质机器机制中的两个关键方面，即亚基之间构象转变的传播和底物被操纵的方式。将在ClpB上进行独特的单分子实验和创新模拟的结合，并将揭示与功能相关的构象变化在ClpB亚基之间的实时传播。机器变构循环期间的功能状态和结构域运动将使用smFRET方法在从微秒到秒的广泛时间范围内进行测量。计算机模拟，使用smFRET导出的距离，将确定与功能状态相关的ClpB构象，并表征它们之间的运动。为此，将采用基于机器学习的粗粒度模拟和分析。本项目还将揭示SP如何通过ClpB管腔转运。当SP与ClpB分子相互作用时，将在单分子水平上实时追踪。新的混合多尺度计算模型将补充实验，并提供关于从无定形聚集体中提取SP的机理及其穿线过程的原子级信息。这些研究将为纳米机器的实验和计算的协同应用提供一个新的框架，并对未来的多项研究产生影响。这一美国和以色列的合作项目得到了美国国家科学基金会和以色列双国科学基金会的支持。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Disassembly of toxic protein aggregates is an essential quality control mechanism that ensures cell viability under stress conditions. This action is performed by ring-shaped AAA+ (ATPases Associated with diverse cellular Activities) biological nanomachines, such as Clp/Hsp100 (ClpB in bacteria or Hsp104 in yeast), which apply mechanical forces to extract protein molecules from aggregates and translocate them through narrow pores to assist their renaturation process. Understanding, at the microscopic level, the coupling between the conformational dynamics of the nanomachine and the mechanisms of substrate protein (SP) threading and disassembly will enable the elucidation of fundamental aspects of critical cellular processes. This project will synergistically combine hybrid multiscale computer simulations, performed in the lab of Prof. Stan at the University of Cincinnati, US, and single-molecule fluorescence resonance energy transfer (smFRET) experiments, performed in the lab of Prof. Haran at the Weizmann Institute, Israel. Increasing the participation of underrepresented minorities in computational sciences is at the center of educational and mentoring activities integrated with the biophysical research in this project. These activities will include outreach at Central State University (CSU), a Historically Black College and University, and research experience opportunities for underrepresented minorities at the University of Cincinnati. Further, science training programs will be offered for summer camp students at the Cincinnati Museum Center. The US-Israel exchange program included in this project will provide interdisciplinary experience and international perspective for students and postdocs. This project will address two key aspects in the mechanism of protein machines, namely the propagation of conformational transitions between subunits and the way substrates are being manipulated. A combination of unique single-molecule experiments and innovative simulations will be performed on ClpB and will reveal the real-time propagation of function-related conformational changes between the subunits of ClpB. Functional states and domain motions during the allosteric cycle of the machine will be measured using smFRET methodology over a broad range of timescales, from microseconds to seconds. Computer simulations, using smFRET-derived distances, will determine ClpB conformations associated with functional states and characterize motions between them. Coarse-grained simulations and analysis based on machine learning will be employed to this end. This project will also reveal how SPs are translocated through the ClpB lumen. SPs will be traced in real time on the single-molecule level as they interact with ClpB molecules. Novel hybrid multiscale computational models will complement the experiments and provide atomistic-level information on the mechanism of extraction of SPs from amorphous aggregates and their threading process. These studies will provide a new framework for the synergistic application of experiments and computations to nanomachines, with implications to multiple future studies.This collaborative US/Israel project is supported by the US National Science Foundation and the Israeli Binational Science Foundation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.