Introduction of a tryptophan reporter group into the ATP binding motif of the Escherichia coli UvrB protein for the study of nucleotide binding and conformational dynamics.

The Journal of Biological Chemistry
E L Hildebrand, L Grossman

Abstract

The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli. This activity is, however, cryptic. Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*). We introduced fluorescent reporter groups (tryptophan replacing Phe47 or Asn51) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function. The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide. Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher. Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the ac...Continue Reading

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Citations

Oct 16, 1999·Proceedings of the National Academy of Sciences of the United States of America·M MachiusJ Deisenhofer
Jul 23, 2015·ACS Chemical Biology·Matthias J KnapeFriedrich W Herberg
Sep 17, 1999·The Journal of Biological Chemistry·E L Hildebrand, L Grossman
Apr 6, 2006·The Journal of Biological Chemistry·Hong WangBennett Van Houten
Jul 17, 1999·Analytical Chemistry·C K LariveS Bogdanowich-Knipp

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