Tryptophan and 8-anilino-1-naphthalenesulfonate compete for binding to trp repressor.
Anilinonaphthalenesulfonate (ANS) and tryptophan compete for binding to the trp repressor protein; thus, the fluorescence decrease associated with ANS dissociation can be used as a fluorometric marker for tryptophan binding to the protein. Using this approach, the tryptophan equilibrium dissociation constant was measured at 25 degrees C to be 3.7 (+/- 1.2) X 10(-5) M, a value which compares favorably with that obtained by other methods for determining the affinity of this ligand. The presence of nonspecific DNA had no effect on the binding affinity, whereas addition of trp operator DNA yielded a 6-fold increase in affinity of tryptophan binding. The kinetics of tryptophan binding to the aporepressor were monitored directly and by ANS displacement at 4 degrees C. The association rate constant was approximately 4 X 10(6) M-1 s-1, and the dissociation rate constant was approximately 60 s-1. The ratio of these values agrees with the binding constant determined by equilibrium dialysis at this temperature. Using the gel retardation method (Carey, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 975-979), the dissociation rate constant for the 40-base pair operator fragment was estimated to be 2 X 10(-2) s-1, which combines with the measured Kd of 0.3 nM to yield an association rate constant comparable to other DNA binding proteins (approximately 10(8) M-1 s-1).
Chou
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WY
Department of Biochemistry, Rice University, Houston, Texas 77251.
Bieber
C
C
Matthews
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KS
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001426:Bacterial Proteins; D001665:Binding Sites; D001667:Binding, Competitive; D004247:DNA / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes; D007700:Kinetics; D007763:Lac Operon / Q000502:physiology; D009875:Operator Regions, Genetic / Q000502:physiology; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*; D029968:Escherichia coli Proteins
D000817:Anilino Naphthalenesulfonates; D001426:Bacterial Proteins; D004247:DNA; D005456:Fluorescent Dyes; D012097:Repressor Proteins; D014157:Transcription Factors; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins; C027132:1-anilino-8-naphthalenesulfonate