The possible contribution of salt bridges and H-bonds, primarily between the gamma subunit and (alpha beta)3 ring, to the generation of torque is the focus of the current project. This is being tested by analysis of site-directed mutations that selectively remove or alter these salt bridges. At the time that the proposal was funded, we anticipated that the determination of the speed of the power stroke under a single set of conditions would be sufficient to determine the torque accurately. We now realize that important controls need to be run to insure that the apparent mutant-dependent changes in power stroke velocity result from normal 120 CCW rotational events. The supplement will accelerate our ability to complete the analyses of these mutants that includes these important control experiments.Aim 3 includes the goal of comparing the power stroke and dwell thermodynamics of wild type and mutants with decreased torque. We completed rrhenius analyses of wild type single molecules. However, our ability to collect data is dependent on the long time required to stabilize temperature at each temperature examined. The supplementary funding will accelerate our ability to complete these experiments.Aim 4 uses molecular dynamic simulations to obtain new insight into the possible rotational trajectory of the gamma-subunit. When the project was originally funded the university did not charge for CPU time. However, the computer cluster used to acquire data has now been retired and the university has now imposed significant charges for CPU time using the new computer. The charges were not budgeted for the current funding cycle. This has contributed significantly to the pay-cuts and temporary lay-offs that I have had to impose to stay on budget. Consequently, an administrative supplement is vital for us to achieve the goals of the project. We anticipate that the simulations will proceed faster with the new computer cluster and the project will be accelerated significantly as a result of the additional funds. The supplementary funds will keep people employed on the project who have extensive expertise and experience that is unique to this project, and without whom progress will slow dramatically. Without funds everyone will be laid off in a few months.
|Effective start/end date||12/28/09 → 11/30/10|
- HHS-NIH: National Institute of General Medical Sciences (NIGMS): $180,202.00
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