FLUORESCENCE ENERGY TRANSFER STUDIES OF PROTEIN INDUCED DNA BENDING

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We have used time-resolved fluorescence energy transfer to investigate protein-induced DNA bending in solution. We are measuring multiple protein-to-DNA and DNA-to-DNA distances in each of two protein-DNA complexes that exhibit dramatic protein-induced DNA bending in the crystalline state: the CAP-DNA complex and the TBP-DNA complex. We are determining DNA bend angles and trajectories in solution, assessing the salt-dependence of the protein-induced DNA bending, assessing the effects of protein mutants on protein-induced DNA bending and assessing the effects of DNA mutants on protein-induced DNA bending. We are using AEDANS ??ex=340nm; ?em=480nm) as the donor fluorochrome and fluorescein (?ex=490 nm; ?em=520 nm) as the acceptor fluorochrome. We have incorporated AEDANS at specific sites in protein by site-directed mutagenesis to introduce unique surface cysteines followed by cysteine-specific chemical modification, and we have incorporated fluorescein at specific sites in DNA by total synthesis. Preliminary results have been reported.