A Bruker ER300-10/12 EPR spectrometer and accessories for carrying out X- and Q-band EPR and saturation transfer (ST-EPR) measurements on biological samples will be purchased. This instrument is ideally suited for spin label and spin probe studies of the structures, dynamics and interactions of soluble and membrane bound proteins, topics of major research emphasis in the laboratories of the above user group. Acquisition of the requested equipment will have an immediate and significant impact on each of the seven projects listed in this proposal. These include: 1) increased access to EPR instrumentation for making routine X-band measurements on biological samples, 2) access to hardware for making higher frequency {O-band) measurements enabling more rigorous analysis of spin labeling data from a number of the studies and detection of protein bound Mn+: in others, 3) capability to make high signal- to-noise ST-EPR measurements in the dispersion mode, 4) access to selected Zeeman-field modulation frequencies for optimizing sensitivity of ST-EPR measurements to changes in the rotational diffusion of a spin labeled protein and for analysis of data from multiple motional populations in ST-EPR experiments, and 5) support for maintenance and repair of the spectrometer and accessories. EPR spectroscopy is currently used by each of the applicants for studies which revolve around a central theme of structure-function relationships of proteins. This group has an established record of design and chemical synthesis of novel spin-label reagents for site-specific modification of proteins and for development of theoretical approaches for analysis of data obtained from well defined systems. The EPR work is complemented by a variety of other biophysical approaches including nuclear magnetic resonance (NMR), X-ray crystallography/molecular modeling, fluorescence spectroscopy, and molecular biology/protein engineering. The applicants have an established record of sharing instrumentation and of active collaborations in their research programs.
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