Regulation of protein expression is crucial to cellular homeostasis and growth, and the noncovalent complexes between proteins and DNA play a fundamental role in the mechanism of regulation. The long- term goal of the research presented here is to acquire a working knowledge of the physical mechanisms underlying the sequence specificity of proteins and other ligands that interact with DNA. A detailed, molecular understanding of the factors important in stabilizing sequence-specific interactions is significant to the development of drugs that interact with DNA, the elucidation and prediction of the binding properties of newly-discovered proteins, to understanding the solution structure, and structural repertoire of the interacting molecules, and to a deeper comprehension of the biochemistry of living systems. Towards this end, the physical chemistry of the interaction of wild-type and mutant forms of EcoRI endonuclease with DNA will be studied by perturbation footprinting (PFP), i.e. quantitative footprinting as a function of pressure, temperature, and ionic strength. The effect of pressure and temperature upon the quaternary structure of EcoRI will be investigated using fluorescence anisotropy. Single base pair- resolution PFP will be performed using the light-activated uranyl ion as the reagent to hydrolyze the phosphodiester bonds exposed to the solvent. The use of high pressure is emphasize in several of the experimental procedures because of its utility for perturbing noncovalent interactions in water. These studies will simultaneously yield data on the thermodynamics and structure of the EcoRI-DNA complex, thereby giving insight in to: 1. the relative energetics of the individual protein-DNA contacts that comprise the complex, 2. the existence of low-lying conformational states of the enzyme, and 3. the structure of these states. The thermodynamic stability and conformation of double-stranded B-form DNA, DNA hairpins, triple helices, and Z DNA will be investigated as a function of pressure, temperature and ionic strength using calorimetry and an adaptation of PFP.
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