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

NSF-BSF：AAA 蛋白质解聚机动力学和功能的协同多尺度建模和单分子荧光研究

基本信息

批准号：
2136816
负责人：
George Stan
金额：
$ 85.15万
依托单位：
University of Cincinnati Main Campus
依托单位国家：
美国
项目类别：
Standard Grant
财政年份：
2021
资助国家：
美国
起止时间：
2021-12-01 至 2025-11-30
项目状态：
未结题

来源：
https://www.nsf.gov/awardsearch/showAward?AWD_ID=2136816&HistoricalAwards=false
关键词：
NSF BSF Synergistic Multiscale Modeling

项目摘要

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.

有毒蛋白质聚集体的分解是确保细胞在应激条件下生存的重要质量控制机制。这一作用是由环形 AAA+（与多种细胞活动相关的 ATP 酶）生物纳米机器执行的，例如 Clp/Hsp100（细菌中的 ClpB 或酵母中的 Hsp104），它们应用机械力从聚集体中提取蛋白质分子，并通过狭窄的通道将其移位。毛孔以协助其复性过程。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。该项目将协同结合美国辛辛那提大学斯坦教授实验室进行的混合多尺度计算机模拟和魏茨曼大学哈兰教授实验室进行的单分子荧光共振能量转移（smFRET）实验研究所，以色列。增加代表性不足的少数群体对计算科学的参与是该项目中与生物物理研究相结合的教育和指导活动的核心。这些活动将包括在中央州立大学（CSU）、一所历史悠久的黑人学院和大学进行外展活动，以及在辛辛那提大学为代表性不足的少数族裔提供研究经验机会。此外，辛辛那提博物馆中心还将为夏令营学生提供科学培训项目。该项目中包含的美国-以色列交流计划将为学生和博士后提供跨学科经验和国际视野。该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的操纵方式。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 ClpB 亚基之间功能相关构象变化的实时传播。将使用 smFRET 方法在从微秒到秒的广泛时间尺度内测量机器变构循环期间的功能状态和域运动。使用 smFRET 导出的距离进行计算机模拟，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。该项目还将揭示 SP 如何通过 ClpB 管腔易位。当 SP 与 ClpB 分子相互作用时，将在单分子水平上实时追踪它们。新颖的混合多尺度计算模型将补充实验，并提供有关从无定形聚集体中提取 SP 的机制及其线程过程的原子级信息。这些研究将为纳米机器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。这个美国/以色列合作项目得到了美国国家科学基金会和以色列两国科学基金会的支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的智力优点和更广泛的影响审查标准进行评估，被认为值得支持。