Theoretical and MD Simulation Studies of U1A-RNA Binding and Specificity

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DESCRIPTION (provided by applicant): The objective of the proposed project is to elucidate the mechanism of U1A protein stem loop RNA binding based on computational models of dynamical structures obtained from molecular dynamics (MD) simulation and corresponding continuum electrostatics and free energy calculations using several methods as a cross check on results. U1A is a prototype of a large class of RNA binding proteins which utilize a characteristic structural motif comprised of an sandwich fold that forms a four-stranded antiparallel -sheet supported by two -helices known as the RNA recognition motif (RRM) or RNA binding domain (RBD). The U1A RNA complexation process appears to be multistep affair in which the outstanding problems are the mechanism of structural adaptation (induced fit or conformational capture), local U1A - RNA contacts vs. cooperative effects in the binding event, solvent release and magnitude of the entropy involved, and the contribution of various chemical forces to binding affinity. Structural adaptation effects will be studied by comparison of the calculated dynamical structures for U1A and RNA free in solution with those of the complexed state. The cooperative networks of protein residues involved in RNA binding will be investigated based on calculated cross correlations of atomic fluctuations from the MD, which reveal through space domain correlations. The link between dynamical structure and functional energetics will be established using an additive free energy component calculation of free energy, enthalpy and entropy resolved into contributions from various amino acids, ribonucleotide bases and into electrostatic, van der Waals repulsions and dispersion terms due to solute and solvation. The proposed research will focus initially on U1A RNA and a set of protein mutants and RNA base replacements in regions critical to observed affinity and specificity and in a later phase be extended to a series of related complexes for which initial structural determination and observed binding affinities and specificities indicate to be critical for a full understanding of RRM/RBD RNA complex formation. The proposed research is closely correlated with concurrent experiment based bio-organic and biophysical chemistry, time resolved fluorescence anisotropy and NMR structure determination on U1A RNA and related systems currently in progress in the Chemistry Department and Molecular Biophysics Training Program at Wesleyan University.