The objectives of this project are to develop and evaluate novel molecular modeling and computational approaches to extract accurate structural information from site-directed spin-labeling (SDSL) studies. The combination of SDSL and electron paramagnetic resonance spectroscopy (EPR) has made remarkable advances in the past 15 years (for reviews see (Hubbell and Altenbach, 1994;Hustedt and Beth, 1999;Hubbell et al., 2000; Mchaourab and Perozo, 2000;Columbus and Hubbell, 2002;Klug and Feix, 2005) and it is now widely employed to study the structures and structural transitions of proteins and large macromolecular assemblies, many of which have been refractory to structural characterization by techniques such as X-ray crystallography and NMR. Even in those cases where atomic resolution structures can be obtained, SDSL is being increasingly employed in complementary studies to elucidate the dynamics of structural transitions that determine biological function (e.g. (Dong et al., 2005). In addition, SDSL has been employed to study the assembly of macromolecular complexes composed of elements whose individual structures have been determined by NMR or X-ray crystallographic methods (e.g. (Park et al., 2006)). We propose to combine the full power of modern molecular dynamics and computational approaches in the Lybrand laboratory with advanced EPR methods in the Hustedt laboratory to develop tools that will dramatically improve the quality of the structural models that are obtained from SDSL data. Molecular dynamics (MD) and Monte Carlo (MC) modeling strategies will be developed to treat the structure and dynamics of the methanethiosulfonate spin label (MTSSL). These strategies will be used to predict both the continuous wave EPR (CW-EPR) lineshapes of singly labeled proteins and the distance distributions measured by both CWEPR and double electron-electron resonance (DEER) experiments on doubly labeled proteins. These new computational algorithms will be tested on the well-studied protein T4 lysozyme (T4L) and then used to investigate the effect of a proline to arginine mutation on the structure and dynamics of the cytoplasmic domain of the erythrocyte anion exchange protein, band 3 (CDB3) in an ongoing collaboration with Project 2 of this grant proposal.. The overall goal of the following Specific Aims is to enable the maximum structural and dynamic information to be derived from a given set of EPR data.
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