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拆卸是一种必不可少的质量控制机制，可确保在应力条件下细胞活力。该动作由环形AAA+（与各种细胞活性相关的ATP酶）进行生物纳米机器，例如CLP/HSP100（细菌中的CLPB或酵母中的HSP104），将机械力施加机械力来从聚集物中提取蛋白质分子，并通过狭窄的孔转移到狭窄的孔中以帮助其恢复过程。在显微镜水平上，了解纳米机械构象动力学与底物蛋白质（SP）螺纹和拆卸机理之间的耦合将使关键细胞过程的基本方面阐明。该项目将协同结合混合多尺度计算机模拟，该模拟在辛辛那提大学的Stan教授和单人分子荧光共振能量传递（SMFRET）实验中进行，在以色列韦斯曼研究所的Haran实验室中进行。增加代表性少数群体参与计算科学的参与是与该项目的生物物理研究集成的教育和指导活动的中心。这些活动将包括在历史悠久的黑人学院和大学的中央州立大学（CSU）的外展活动，以及辛辛那提大学代表性不足的少数民族的研究经验。此外，将为辛辛那提博物馆中心的夏令营学生提供科学培训计划。该项目中包括的美国 - 以色列交流计划将为学生和博士后提供跨学科的经验和国际观点。该项目将解决蛋白质机械机理的两个关键方面，即亚基和底物操纵底物之间的构象转变的传播。将在CLPB上进行独特的单分子实验和创新模拟的组合，并将揭示CLPB亚基之间与功能相关的构象变化的实时传播。机器变构循环期间的功能状态和域运动将在从微秒到秒的各个时间范围内使用SMFRET方法进行测量。使用SMFret衍生距离的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动作。基于机器学习的粗粒模拟和分析将用于此目的。该项目还将揭示如何通过CLPB管道易位的SP。当SP与CLPB分子相互作用时，SP将在单分子水平上实时追踪。新型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提取SP的机理及其螺纹过程的原子级别的信息。这些研究将为实验和计算在纳米机器上的协同应用提供一个新的框架，对以后的多项研究产生影响。美国国家科学基金会和以色列国家科学基金会和以色列基础科学基金会的支持。该奖项反映了NSF的法定任务，并通过评估范围来反映了支持者的知识群体的支持和宽广的基金会。