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J. Biol. Chem., Vol. 269, Issue 11, 7919-7925, Mar, 1994

Introduction of a tryptophan reporter group into loop 1 of the recA protein. Examination of the conformational states of the recA-ssDNA complex by fluorescence spectroscopy

E Stole and FR Bryant
Department of Biochemistry, Johns Hopkins University, School of Public Health, Baltimore, Maryland 21205.

Site-directed mutagenesis was used to replace His-163 in the Loop 1 region of the recA protein with a tryptophan residue. The [H163W]recA protein binds single-stranded DNA (ssDNA), catalyzes ssDNA-dependent ATP hydrolysis, and is fully active in the three-strand exchange reaction. In addition, the fluorescence properties of the Trp-163 reporter group are very sensitive to the binding of nucleotide cofactors to the H163W]recA-ssDNA complex. The fluorescence of Trp-163 is modestly quenched by the binding of ADP (21%) and strongly quenched by the nonhydrolyzable ATP analog, ATP gamma S (70%); since ADP and ATP gamma S stabilize the closed and open conformations of the recA-ssDNA complex, respectively, the quenched states observed with these nucleotides likely reflect differences in the fluorescence properties of tryptophan 163 in these two states. ATP has a more complex time- dependent effect on Trp-163 fluorescence. When ATP is added to [H163W]recA-ssDNA complexes, there is an immediate quenching of Trp-163 fluorescence (44%) which is intermediate in intensity between that observed with ADP and ATP gamma S. The ATP-induced quenching gradually decreases with time as the pool of ATP is converted to ADP by the ATP hydrolysis activity of the [H163W]recA protein. These results are discussed with regard to the nucleotide cofactor-dependent conformational transitions of the recA-ssDNA complex.
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