Comprehensive experimental information on the essential contributions of intramolecular dynamics to 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 and ligand recognition;and for design of novel proteins and protein ligands, including pharmaceutical agents. This research project will use multidimensional NMR spectroscopy to address these fundamental issues. One long-term goal is to define the molecular determinants of stability and catalytic activity of the enzyme 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. The enzyme is distributed widely in prokaryotes and eukaryotes, and retroviral reverse transcriptase contains a C-terminal RNase H domain. Another long-term goal is to define the molecular determinants of ligand binding, including aspects of specificity, and allosterism, in DNA recognition by the yeast protein GCN4 and in nucleotide binding by the ATP-binding cassette (ABC) MJ1267 of the branched chain amino acid (LIV) transporter from Methanococcus jannaschii. GCN4 is the prototypical member of the bZip family of transcription activators. 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 understanding normal biological function and pathology. GCN4 represents an example of induced fit molecular recognition through a disorder-order transition associated with DNA binding. The LIV transporter is a member of a diverse family of ABC transporters involved in numerous biological processes and diseases, including multidrug resistance and cystic fibrosis. ABC MJ1267 represents an example of selected-fit molecular recognition and allosteric transmission of conformational changes between remote sites in proteins.
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