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 have a long-term program of research to characterize dynamics underlying a number of enzymatic and protein-protein interactions. This has included the development of protein engineering methods including segmental labeling for NMR, the direct determination of interdomain orientations in solution from NMR relaxation, in cell studies of protein-protein binding, and direct, and complete detection of protein-protein interaction surfaces. ESR techniques are generally required to augment the experimental input for modeling structures and dynamics by providing distances (larger) and time scales (shorter) than available from NMR methods. Protein tyrosine kinases(PTKs): We are concentrating on two aspects of structure/dynamics ?(a) the role(s) of activation loop phosphorylation in enhanced activity of PTKs, and (b) the role(s) of protein-protein interactions via intramolecular/ interdomain and intermolecular allosteric modulation of activities and localization. The non-receptor Csk, Src, Abl , and receptor PTK FGFR-2 are currently expressed and being studied in multiple engineered forms with both natural, frequently disease ?related, and other mutations/isoforms/truncations. We are extending these studies to incorporation into eukaryotes, and ESR potentially provides a sufficient sensitivity to overcome the issue of whether excessive protein incorporation is needed to see the cargo modification of NUP/karopherin affinities.
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