EXCEED THE SPACE PROVIDED. Protein recognition of specific DNA sequences lies at the heart of many normal and disease-related processes, including gene expression and its regulation. This study focuses on adducing general principles applicable to any site-specific protein-DNA interaction, by linking structural information, thermodynamics and dynamic behavior. Major attention will be given to dynamic characteristics of protein-DNA complexes, including binding-dependent conformational transitions in proteins and DNA and conformational-vibrational fluctuations in the complexes. Five-year aims include: 1. Continuing studies on the relationship between molecular strain and thermodynamics of protein-DNA interactions, specifically: (a) Determining the contributions to electrostatic strain, to binding free energy (AG?bjnd) and to heat capacity change (AC?p), of each residue of the acidic cluster in the BamHl active site; (b) Using these data on mutant and wild-type enzymes to benchmark computational modeling of the electrostatics and dynamics of these complexes; (c) Studying the role of asymmetric charge neutralization in DNA bending by EcoRV endonuclease, using steady-state and time-resolved Foerster resonance energy transfer (trFRET) spectroscopy and 2D pulsed FT-ESR.. 2. Continuing studies of the molecular basis of relaxed-specificity mutations in EcoRI endonuclease by (a) Testing the hypothesis that 'promiscuous' mutant EcoRI endonucleases form 'specific'-like complexes at incorrect DNA sites, using steady-state FRET and FT-ESR. (b) Testing the hypothesis that these 'promiscuous' mutations have in common altered dynamics of the EcoRI endonuclease 'arms', by trFRET spectroscopy and 2D-FT-ESR spectroscopy techniques, (c) Determining the binding and cleavage parameters and thermodynamic parameters (AH?, AS0, AC?p) for proteins containing both a 'promiscuous' mutation and a second suppressor mutation 3. Further investigation of how context surrounding the DNA recognition site modulates the specificities of restriction endonucleases by (a) Testing the hypothesis that the dynamics of the protein backbone are influenced by context changes that affect AG0bind and AC?p, using NMR relaxation parameters, their temperature-dependence and hydrogen- exchange studies to test the inference that molecular strain broadens the distribution among configurational microstates. (b) Determining how DNA backbone modifications that relieve sensitivity to sequence context affect the AC?p for endonuclease-DNA association, as assessed by ITC and van't Hoff analysis.
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