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 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 active UvrB* form (Kd of approximately 1 mM). The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.
Hildebrand
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Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
Grossman
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D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004265:DNA Helicases; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009711:Nucleotides / Q000378:metabolism*; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D009711:Nucleotides; C058093:UvrB protein, E coli; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004265:DNA Helicases