PROJECT SUMMARY Conformational changes of proteins are required for nearly all biological functions and inappropriate conformational transitions are associated with numerous pathologies, including gain or loss of function mutations, misfolding diseases, and pathogen drug resistance mutations. Comprehensive experimental information on intramolecular dynamics and intermolecular kinetics is critical for biophysical understanding of structure/function relationships; for mechanistic interpretations of kinetic processes, such as enzyme catalysis and ligand recognition; for understanding “action at a distance” in allostery or regulation of activity; and for design of novel proteins and protein ligands, including pharmaceutical agents. Conformational changes in proteins, including local librations, loop and hinge motions, relative motions between domains, collective “breathing” of protein cores, ligand-binding or oligomerization reactions, and overall folding-unfolding events, may be closely coupled, and in some instances rate-limiting, to biological functions such as molecular recognition, transitions along the catalytic cycle of enzymes, and inhibition or activation of proteins through intra- or inter-molecular protein-protein interactions. Recent developments, including those from the PI laboratory, have opened new opportunities for investigation of conformational dynamic processes using NMR spin relaxation measurements (and other NMR observables) at equilibrium in solution and with atomic site resolution, without potential complications introduced by non-native modifications necessary for other solution- state spectroscopic techniques. In addition, close coupling between experimental measurements and molecular dynamics (MD) simulations or other theoretical approaches allow feedback with experiment in interpreting results, formulating hypotheses for on-going investigation, and improving both experimental and theoretical techniques. The present proposal will use NMR spectroscopic and computational approaches to explicate the functional roles of conformational transitions in enzyme catalysis, including ribonuclease HI (and other members of the nucleotidyl-transferase superfamily) and the DNA-repair protein AlkB; in DNA recognition by transcription factors, including Hox and bZip proteins; and in protein-protein molecular recognition interactions, including strand-swapping and dimerization by cadherin cell-adhesion proteins. The biological goals are based on recent advances in the PI laboratory (i) establishing a quantitative relationship between active-site loop dynamics and enzyme activity for ribonuclease HI homologs, (ii) describing a multi-state coupled folding and binding mechanism for dimerization of cadherin proteins, and (iii) identifying both pre- formed and induced conformations of transcription factors and target DNA in tuning selectivity and affinity. Completion of these goals will provide exquisite insight into strucuture/dynamics/functiona...