We propose to use time-resolved fluorescence energy transfer to investigate protein-induced DNA bending in solution. We will measure 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 will determine DNA bend angles and trajectories in solution, assess the salt-dependence of the protein-induced DNA bending, assess the effects of protein mutants on protein-induced DNA bending, assess the effects of DNA mutants on protein-induced DNA bending. We have chosen AEDANS (lex=340nm; lem=480nm) as the donor fluorochrome and fluorescein (lex=490 nm; lem=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. Pilot experiments support the feasibility of the proposed work but indicate the need for a laser system with a more stable baseline and with subnanosecond time resolution.
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