THE DYNAMICS AND PATHOLOGIES OF MOLECULAR MOTORS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The realization that many essential functions of living cells are performed by nanoscale motors consisting of protein complexes has given rise to an intense effort to understand their mechanisms. We focus on F1 ATPase and DNA polymerase I, two very different molecular motors of fundamental importance in biology. We propose to perform atomic-scale simulations to obtain information not available from experiment. The research will lead to a deeper understanding of the function of these motors and in the case of DNA polymerase, show how malfunction is prevented. F1 ATPase, the smallest biological rotary motor, is composed of seven units, six of which (alpha3beta3) form a spherical globular construct around a central shaft, the gamma subunit, which rotates 1000/sec as a result of ATP hydrolysis in the catalytic beta subunits; when an applied torque rotates the g subunit in the reverse direction, the motor synthesizes ATP, its normal function in the cell. Our previous studies investigated the pathway of the conformational change and the nature of the mechanical coupling. The essential next step is to evaluate the coupling between the chemical steps (hydrolysis or synthesis of ATP) and the subunit conformations on the rotational pathway. Free energy simulations will be used to find the conformations that favor hydrolysis or synthesis. That such conformations exist is an essential aspect of this remarkable motor, which makes possible efficient synthesis or hydrolysis of ATP, depending on the direction of the rotation of the g subunit. Given these conformations, combined quantum mechanical/molecular mechanical simulations will be performed to evaluate the free energy barrier of the reaction and elucidate the origin of the catalytic rate enhancement. DNA polymerases are responsible for the accurate copying of genetic information from one cell generation to the next. Our previous studies have determined the details of the translocation step, an essential part of the motor function. It occurs after the addition of a base to the primer strand, so as to position the polymerase on the DNA for adding the next base. The results of this analysis will make possible exploration of the mechanism by which mismatches in DNA (i.e., critical errors that can cause cancer) stall DNA replication, a mechanism by which the essential high fidelity is achieved. Known crystal structures of the polymerase I bound to DNA with mismatched bases make possible simulations to determine the effect of these on the translocation step. In addition, based on a new single molecule experiment, the effect of mismatches on slowing the fingers closing transition will be explored. Finally, again using known crystal structures, free energy simulations will be performed to determine the effect of mismatches on the configuration of the active site. The results will complement our analysis of normal DNA replication by providing an understanding of certain pathologies. This is of considerable medical importance, as well being of interest itself and a subject of intense experimental research. Two very different, but important motors are included in the same proposal because the complementarity of the research will make the results all the more meaningful. The research in both areas requires multiple closely related simulations, which can be done most efficiently in parallel. Moreover, since specific details of subsequent calculations depend critically on the previous results, it is essential also to perform these simulations rapidly with fast turn-around. Given the large size of the systems under investigation (on the order of 180,000 particles for F1 ATPase and 140,000 for DNA polymerase I complex), a state-of-the-art supercomputer will be essential not only for large-scale production following the standard paradigm, but also as a research tool intimately coupled to the computational design.