Comprehensive experimental information on the essential contributions of intramolecular dynamics to the biological functions of proteins is critical for biophysical theories of equilibrium properties, such as heat capacity and thermal stability; for mechanistic interpretations of kinetic processes, such as enzyme catalysis, ligand recognition, and protein folding for the de novo synthesis of proteins; and for design of novel protein ligands, including pharmaceutical agents. This research project will use modern multidimensional NMR spectroscopy and nuclear spin relaxation measurements to characterize the conformational dynamics of proteins in order to address these fundamental issues. One long-term goal of this research is to define the molecular determinants of stability and biological activity of ribonuclease HI (RNase H) by comparing the structural, dynamical and enzymatic properties of homologous proteins derived from Escherichia coli and the extremely thermophilic bacterium Thermus thermophilus. In particular, the temperature dependence of conserved structural and dynamical features of E. coli and T. thermophilus RNase H will be used to elucidate thermodynamic and kinetic principles governing protein structure and function. RNase H is an endonucleases that hydrolyzes the RNA moiety in RNA-DNA hybrid oligonucleotides. The enzyme is distributed widely in prokaryotes and eukaryotes, and retroviral reverse transcriptase contains a C-terminal RNase H domain. RNase H is a potential target for anti-retroviral drugs and is an important component of antisense oligonucleotide therapeutic approaches. Another long-term goal of this research is to characterize configurational entropic contributions to DNA recognition by the yeast protein GCN4, the prototypical member of the bZip family of transcription activators, by comparing dynamic and thermodynamic properties of mutant protein motifs and variant target DNA sequences. The DNA-recognition helices in the bZip motif are stable only in the presence of DNA. The role of individual amino acids and nascent structures in modulating the entropic effects of the disorder-order transition associated with DNA binding will be delineated. Motifs that recognize specific DNA sequences are ubiquitous components of proteins that regulate gene expression; consequently, explication of the molecular basis for recognition is critical for development of a molecular theory of normal biological function and pathology.
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