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. PDS conducted on spin-labeled proteins at low temperatures yields distance distributions. However, it is not well understood to what extent protein conformations represented by these distributions correspond to conformations in solution. In general there is an argument that the structure determined at such conditions may be a poor model for highly dynamic (flexible) proteins. Similar arguments, such as protein confinement by the crystal lattice and using cryogenic temperatures for collecting diffraction pattern could be (and are) applied to X-ray crystallography. But we do know that there is often significant correspondence between X-ray and NMR derived structures. Therefore it is important to test what the distance distributions from PDS conducted on proteins in solution in the absence of confinement by crystal contacts may represent. One argument is that the range of conformations given by the distribution of end-to-end distances depends mainly on spring constants and the number of connecting chemical bonds. One would expect to find this for example for a rod like organic molecule or long alpha-helix, however one would need to take a sufficiently long helix in order be able determine the extent of its flexibility and may also need to use conformationally-persistent spin-labels, such as TOAC. This was the approach used in our collaborative paper with D. Budil et al. However, for a folded protein or a protein complex the picture may be more complex, since many bonds allow for substantial freedom and may produce an ensemble of conformations that exchange slow and can get trapped. Therefore, sufficiently rapid freezing that brings the solvent into the glassy state as a matter of microseconds is expected to preserve the ensemble of such conformers, but may depopulate those that exchange with faster rates in microsecond range. We are going to apply freeze-quenching technique, developed in Scholes lab, using different freezing rates on doubly labeled model proteins and to compare distances distributions obtained by PDS with the goals to delineate conformational exchange rates and to test the relevance of distance distributions from PDS to the range of conformations sampled by a protein in solution. .
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