This research project is designed to investigate the mechanisms by which proteins recognize specific sequences of DNA and the means by which small ligands modulate the recognition process. The lac and trp repressors from E. coli represent regulatory proteins in the classes of inducible and repressible systems; both of these proteins and their target DNA sequences can be isolated in the quantities required for physical and chemical studies, and the coding regions are available on vectors for site-specific mutagenesis and other genetic manipulations. The specific objectives are: (1) assessment of the roles of specific amino acids and regions of the primary sequences in the function of these repressor proteins. This examination will include generation of site-specific mutants based on the trp repressor x-ray crystallographic structure and on the known chemical, physical and homology data for lac repressor. Interchange of the helix-turn-helix structure implicated in DNA binding between the two repressors will be undertaken. These specifically designed proteins and mutant proteins available from other sources will be characterized in detail. Antibodies to specific segments of the primary sequence of these proteins will be generated, and the effects of antibody-repressor complex formation on functional activities will be measured. (2) Determination of the effects of DNA structure on binding activities. The effects of supercoiling on the binding parameters for repressor-DNA (both operator and nonspecific binding) will be examined. Cross-linking of proteins to DNA will be attempted to assess specific regions of the protein which are in close proximity to DNA. (3) Thermodynamic studies of protein structure and function. High pressure dissociation of the repressor proteins and effects of ligands on this process will provide information about the strength of inter-subunit contacts and alterations in these contacts by ligand binding. Calorimetric measurements of protein denaturation (DSC) and ligand binding will be undertaken to assess the thermodynamic properties of the proteins and their interactions. (4) Determination of protein structure and conformational changes. FLuorescence spectroscopy of intrinsic and extrinsic fluorophores will be measured, and NMR spectroscopy will be utilized to examine the binding properties of the trp repressor. The correlation of data obtained from these studies on two different systems with data available in the literature will expand our knowledge of genetic regulation, an essential function in all living organisms.
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