Protein DNA Specificity Determinants by Paramagnetic Metal-Based ESR Distance Measurements

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Intellectual Merit: There are two major accomplishments associated with this project: (a) Developing copper-based pulsed ESR experiments with the aim of generalizing the technique to proteins that do not normally bind metals, achieving up to a three-fold enhancement in signal-to-noise ratio through double quantum coherence (DQC) methods for paramagnetic metal ions and exploiting such techniques to learn about the packing of the side chain that is typically used to spin label proteins. The distance measurement methodology will have a significant impact on the ability to understand structure-function relationships in proteins that cannot be studied by traditional biophysical methods. A series of distance constraints between copper(II) centers site-selectively labeled on a protein, X-ray structures and molecular modeling will provide detailed insights into the packing and flexibility of side chains used to place paramagnetic metal ions at selected sites in proteins. Such information will be especially important for paramagnetic relaxation enhancement based structure determinations in NMR. (b) Probe the relationships among structure, protein dynamics, and thermodynamics in the restriction endonuclease EcoRI to adduce general principles of site-specific protein-DNA recognition. Pulse-ESR distance methods (both DEER and DQC) and X-band and W-band continuous wave ESR spectroscopy will be applied to study the differences in dynamics and distance distributions between complexes of EcoRI endonuclease bound to specific, miscognate (one incorrect base pair) and nonspecific DNA sites. A newly acquired W-band ESR spectrometer greatly enhances the ability to distinguish protein backbone dynamics from motions of the spin label and provides the exciting opportunity to characterize motions on the sub-nanosecond timescale. This will be one of the first examinations of how ESR-based dynamic parameters differ not only at different locations within a macromolecular complex, but also at corresponding locations in a set of related recognition complexes. Such comparative measurements will provide information on how microscopic differences in structure and dynamics contribute to macroscopic differences in thermodynamic parameters. Broader Impact:Six to eight undergraduate researchers and two to three graduate students working on this project will be trained at the interdisciplinary interface between chemistry and biophysics. The PI will seek to recruit undergraduates from 4-year colleges by actively presenting research at those institutes. Workshops will be organized for high-school teachers, in the Pittsburgh area, to orient them with this research as well as provide modules for use in high school curricula. Workshops for high school students will expose them to careers in Chemistry. Software for the extraction of distances from metal-based ESR data will be disseminated. Two women will be trained in ESR as part of the support from this project. Research ideas and themes developed in the project will be used to modernize the undergraduate Physical Chemistry course. This initiative is in line with the American Chemical Society's call for the incorporation of interdisciplinary and integrative trends of chemical research into undergraduate curricula without the creation of new course work.