Theoretical and computational investigations into structure-function relationships of biomolecular machines at atomic resolution

Molecular motor;Free energy landscape;Rate calculations;Transition pathway;Molecular dynamics;Multiscale modeling;Enhanced sampling methods;Helicase;ATPase

Ma, Wen

关键词: Molecular motor;    Free energy landscape;    Rate calculations;    Transition pathway;    Molecular dynamics;    Multiscale modeling;    Enhanced sampling methods;    Helicase;    ATPase;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/98292/MA-DISSERTATION-2017.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Molecular machines play vital roles in many cellular processes, including DNA replication and repair, gene expression, protein degradation, protein secretion and maintenance of pH homeostasis. Despite intense research efforts, the atomic-level mechanism transmitting other form of energy into mechanical force is still unclear. Molecular dynamics simulations, though capable of providing atomic details, are limited in the study of molecular machines owing to the challenge that these machine usually function on a millisecond (or longer) time scale which, for a long time, could not be covered computationally. Employing advanced sampling techniques and theoretical modeling, we investigate the mechanism of three exemplary molecular machines in the following. Transcription termination factor Rho is a key factor in bacterial gene expression and regulation. An essential question is how RNA is translocated by Rho using energy from ATP hydrolysis. By combining MD simulations with path sampling techniques and milestoning analysis, we find that the release of hydrolysis product (ADP+Pi) triggers the force-generating process of Rho through a 0.1 millisecond-long conformational transition. Our results not only reveal in new detail the mechanism employed by ring-shaped ATPase motors, for example the use of loosely bound and tightly bound hydrolysis reactant and product states to coordinate motor action, but also provide an effective approach to identify allosteric sites of multimeric enzymes in general. Following the footstep of the study on Rho, we investigate a DNA helicase UvrD, which plays key roles in DNA replication and repair, by unwinding nucleic acid strands. Combining bioinformatics approaches and free energy calculations, we characterize how the UvrD helicase changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The obtained transition pathway shows that UvrD opens the interface between the 2B/1B domains, allowing the bound ssDNA strand to escape and the other strand to bind to the ssDNA-binding domains. An interesting “tilted” conformation is revealed, which serves as a key metastable state for the ssDNA strand exchange. The simulation results not only match the single-molecule measurements from our collaborators, but also decipher key elements for the “hyper-helicase” behavior induced by a mutant (UvrD303). 
The last project is a study of the growth of flagellum, which is an hours-long process. The building blocks of flagella, flagellin monomers, are pumped by a type III secretion system, through the flagellar interior channel to the growing tip. After a flagellin monomer binds to the tip of the filament, the growing flagellum is extended. The flagellin translocation process, due to the flagellum maximum length of 20 μm, is an extreme example of protein transport through channels. By deriving a theoretical model complemented by molecular dynamics simulations, we explain why the growth rate of flagellar filaments decays exponentially with filament length and why flagellum growth ceases at a certain maximum length.