SIMULATING DYNAMICS AND ORIENTATIONS OF ENGINEERED SIDE CHAINS IN THE RESTRICTI

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We request computing time and storage space on TeraGrid resources, specifically Pople, to run extensive parallel molecular dynamics simulations to determine the preferred orientations of a modified side chain of the restriction endonuclease EcoRI. EcoRI exhibits an extremely high binding specificity to a six base pair sequence of DNA, the structural origin of which was investigated using Electron Spin Resonance (ESR). In conjunction with site-directed spin labeling, which introduces a side chain containing a nitroxide label into the protein, distance measurements were performed on EcoRI to provide structural constraints of the system. In order to paint a more accurate picture of the structure and dynamics of the spin labeled locations of EcoRI from ESR measurements, knowledge of the average nitroxide side chain orientation is required. Initial attempts to model the nitroxide side chain using Monte Carlo rotamer search methods in combination with all-atom molecular dynamics have revealed discrepancies between experiment and simulation due to sampling restrictions. This request aims to establish a procedure where extensive MD simulations can be used to reproduce the distance between two nitroxide side chains, enabling the extraction of the C?-C? distance and distribution. The simulations will also allow a discrimination of the role of nitroxide side chain dynamics versus backbone dynamics on ESR lineshapes and distance distributions. We will run 10 parallel simulations at six different sites in the protein using two independent nitroxide force fields. By doubly labeling the crystal structure a total of 60 simulations will be run for 100 ns each to simulate the average nitroxide orientation and dynamics at different secondary structures in addition to investigating the effects of nitroxide parameterization on these observables. Our request of 480K CPU hours will provide the computational power needed to perform long runs that enable sufficient sampling of the nitroxide side chain. In this way, we will work towards our goal of developing an MD simulation procedure that can be applied to different biological systems to reliably model nitroxide side chain orientations and dynamics so that more atomistic details of the protein or DNA structure and dynamics can be better understood.