The objective of the proposed research is to understand the mechanics of macromolecular recognition with emphasis on DNA-protein interactions. Fluorescence Spectroscopy offers the sensitivity, selectivity, and dynamic range to probe changes in structure and dynamics of macromolecular complexes. Steady-state and time-resolved fluorescence techniques will be used to determine environment, solvent accessibility, distances, and motions in individual molecules and in complexes. Three projects address fundamental questions about recognition processes in DNA and proteins. Strategies developed in previous work will be applied to carefully selected model systems. Changes in solution conformation and dynamics involved in sequence-specific recognition of DNA by a protein will be studied using EcoRI endonuclease and its recognition site. The structure of the N-terminal region of the endonuclease, which is essential for DNA cleavage, will be deduced from fluorescence depolarization, lifetime, and energy transfer studies of fluorescent labeled EcoRI. The N-terminal region is not resolved in the EcoRI-DNA cocrystal structure. Local structure and flexibility of the DNA site will be monitored in EcoRl complexes with fluorescent labeled oligonucleotides. These oligonucleotides were previously used in thermodynamic studies to identify critical recognition elements on the DNA. The fluorescence results will probe subtle changes in DNA predicted by the thermodynamic data. Protein conformation changes associated with recognition of a nucleoside will be quantified in single tryptophan mutants of human adenosine deaminase. Amino acids in the environment that affect the fluorescence will be identified based on recent work on tryptophan photophysics. Ground- and transition-state analog inhibitors will be used to trap the enzyme at different stages in the catalytic cycle. Local changes in structure and dynamics will be deduced from the tryptophan fluorescence decays. Global volume changes will be determined from the pressure dependence of nucleoside binding. The relationship between structure and dynamics of DNA will be investigated by time-resolved fluorescence depolarization experiments using a covalently bound probe in the DNA minor groove. The torsional and bending rigidities of homopurine-homopyrimidine and alternating purine-pyrimidine B-DNAs will be determined. Two unusual DNA structures, Z DNA and triple helix, which are thought to be important in gene regulation will also be studied. The torsional and bending rigidities are fundamental mechanical properties of DNA with implications for DNA-protein interactions.
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