We propose to continue our studies of protein polymorphism in virus particles and to extend these to the dynamic analysis of quaternary structure transitions amenable to investigation by time-resolved methods. Our goal is to characterize the tertiary structures of the subunits and to identify the molecular switches and chemical surface features that make them suited for multiple quaternary structure conformations. In each case we have or are determining the near-atomic resolution structure of at least one form of the particle by x-ray crystallography. If alternate polymorphs can not be crystallized because they are moderately heterogeneous or unstable, the atomic subunit model derived from the high resolution structure will be used to develop pseudo atomic resolution structures of the polymorphs with methods described below. All of the systems that we are investigating can be assembled either in vitro or as a recombinant assembly (virus-like-particles; VLPs) within the cells of the heterologus expression system used. Thus our hypotheses from detailed modeling and physical studies can be directly tested by molecular genetic alterations and the analysis of the consequent assembly phenotype. The study of static polymorphs of viral subunits from alfalfa mosaic virus (AMV), cowpea chlorotic mottle virus (CCMV), rice yellow mottle virus (RYMV), flock house virus (FHV), Nudaurelia capensis omega virus (NomegaV) and LA virus will be initiated or extended by a variety of methods. These methods include (a) conventional or ultra-low resolution x-ray crystallography, (b) cryo-electron microscopy and image reconstruction, (c) molecular modeling with high resolution coordinates into low resolution experimental density functions using texture surface mapping and a generalized symmetry server, (d) refinement of derived models by molecular mechanics and energy minimization, (e) solution x-ray scattering and data analysis with icosahedral or spherical harmonics, and (f) proteolytic susceptibility studies with resulting polypeptides analyzed by mass spectrometry. Systems amenable to time resolved analysis include the reversible swelling of CCMV and RYMV and the large scale quaternary structure transition in NomegaV. These will be investigated by time resolved solution x-ray scattering (100 millisecond regime) and cryoEM (500millisecond regime). The long-term goal of these studies is to find the means to interfere with these transitions through the use of rationally designed small molecules. We have chosen the model systems described for their experimental accessibility, but anticipate that their study, in the context described, will provide a general and basic understanding of the principles of protein polymorphism and the ability to specifically alter such transitions.

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National Institute of General Medical Sciences (NIGMS)
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Biophysical Chemistry Study Section (BBCB)
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Scripps Research Institute
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