Characterization of the tryptophan binding site of Escherichia coli tryptophan holorepressor by phosphorescence and optical detection of magnetic resonance of a tryptophan-free mutant.
The L-tryptophan binding site of the Escherichia coli tryptophan holorepressor (trpR) is characterized by low-temperature phosphorescence and optical detection of magnetic resonance (ODMR) spectroscopy. Measurements are made on a tryptophan-free mutant of trpR, W19/99F, in which both intrinsic tryptophan residues of apo-trpR have been replaced with phenylalanine. Thus, essentially all of the phosphorescence that is observed from trpR originates from the bound L-tryptophan corepressor. The phosphorescence and ODMR results for the bound corepressor agree quite well with those obtained previously for the corepressor site in both single tryptophan-containing mutants, W19F and W99F [Burns, L.E., & Maki, A.H. (1994) J. Fluorescence 4, 217-226]. A red shift of the L-tryptophan phosphorescence origin as well as a decrease in the D-E ODMR frequency result from an increase in the local polarizability upon binding at the corepressor binding site. A large decrease in the ODMR line widths signals a reduction of local heterogeneity upon binding. Subsequent binding of trpR to a self-complementary DNA sequence that mimics the trp operator, 5'-CGTACTAGTTAACTAGTACG-3', produces a further decrease in line widths and additional changes in the ODMR frequencies, attributable to an increase in both the D and E parameters. This result demonstrates that binding of holo-trpR to the operator affects the local environment of the bound corepressor.
Li
Z
Z
Department of Chemistry, University of California, Davis 95616, USA.
Maki
A H
AH
Eftink
M R
MR
Mann
C J
CJ
Matthews
C R
CR
D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008163:Luminescent Measurements; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009154:Mutation; D055095:Optics and Photonics; D012097:Repressor Proteins / Q000378:metabolism*; D014364:Tryptophan / Q000378:metabolism*; D029968:Escherichia coli Proteins
D001426:Bacterial Proteins; D012097:Repressor Proteins; D014364:Tryptophan; C027190:TRPR protein, E coli; D029968:Escherichia coli Proteins