The objectives of this project are to develop computational tools that can be used to extract structure and dynamics information from site-directed spin-labeling (SDSL) studies of proteins. These novel tools will have an immediate and significant impact by extending the capabilities of SDSL and electron paramagnetic resonance (EPR) spectroscopy for the determination of protein structures and functional dynamics. Procedures will be developed for calculating a continuous wave EPR (CW-EPR) spectrum directly from molecular dynamics (MD) simulations of a spin-labeled protein. Comparison of calculated lineshapes with experimental spectra of T4 lysozyme (T4L) spin-labeled in a set of different environments will allow a detailed evaluation of how well MD simulations reproduce the dynamic behavior of the nitroxide side chain. A combination of MD and Monte Carlo (MC) modeling strategies will be developed to predict distance distributions between pairs of dipolar coupled spin labels measured by either CW-EPR or double electron-electron resonance (DEER) spectroscopy. The use of low microwave frequency (L-band) and multifrequency (L-, X-, Q-, and W-bands) measurements for distance determination by CW-EPR will be evaluated. Low frequency measurements hold the promise of reducing the influence of the relative orientation of the two nitroxides, thus giving a more pure measure of the distance distribution itself. An alternate spin label with reduced rotational freedom will be evaluated for its utility for both CW-EPR and DEER distance measurements. Finally, the knowledge gained by these efforts will be applied in a SDSL study of the effect of a naturally occurring proline to arginine mutation at residue 327 on the structure of the cytoplasmic domain of band 3 protein (CDB3). This mutation results in hereditary spherocytosis and hemolytic anemia in vivo. The study of the P327R mutation of CDB3 will lead to a better understanding of the architecture of the red blood cell membrane and the role that mutations in band 3 play in hereditary diseases of the red blood cell membrane. In general, the tools developed in this proposal will be applicable to a wide range of SDSL studies of protein structure, assembly, and functional dynamics in laboratories worldwide. ? ? ?
| DeSensi, Susan C; Rangel, David P; Beth, Albert H et al. (2008) Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations. Biophys J 94:3798-809 |
