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

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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.