SUBUNIT INTERACTIONS IN DNA BINDING PROTEINS

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Proteins that bind to nucleic acids are involved in many important biological processes, including replication, transcription, translation, and recombination events. Their affinity for nucleic acids often depends upon levels of certain metabolites, specific ions, and salt concentration. While the binding of a number of DNA binding proteins to their specific recognition sequences has been well characterized, the aggregation state of these proteins has not been the object of systematic investigation. The proposed research focuses on three model protein systems which recognize specific sequences of double stranded deoxyribonucleic acids. Two of these proteins are repressor proteins, the lac and trp repressors. Both are oligomers (tetramer and dimer, respectively) at micromolar concentrations. The third system is the restriction endonuclease, EcoRI, which can form both tetramers and dimers. By determining the free energy of subunit association for these oligomer subunits under varying conditions of ion and metabolite concentration, we will determine if and how interactions at the subunit interface are involved in regulation the proteins' affinity for DNA. The equilibrium methods employed in these studies will be those of high hydrostatic pressure and temperature variations coupled to high sensitivity multi-frequency and steady state fluorometry. High hydrostatic pressure is known to destabilize many protein subunit interactions. Thus, high pressure fluorescence studies of these oligomers labelled with long-life- time fluorescent probes will allow for the determination of the effects of ligation by effector molecules (corepressors, anti- inducers, and inducers), DNA binding, and ionic strength upon subunit associations under equilibrium conditions using nanomolar protein concentrations. Additionally, studies of the intrinsic tryptophan fluorescence polarization and lifetime, as a function of temperature, pressure, and ligation state will yield information as to the changes in protein conformation and dynamics associated with the binding of effector molecules and DNA sequences. This dynamic and thermodynamic data will add to our understanding of the physical mechanisms of the transfer of binding information from one subunit to the others in these biologically important oligomeric proteins.