The allosteric interaction between D-galactose and the Escherichia coli galactose repressor protein.
The Escherichia coli galactose repressor protein (GalR) inhibits transcription of the gal operon upon binding to two operator sites (1-7). This DNA binding activity is inhibited when D-galactose or D-fucose binds to GalR (8-14). Fluorescence spectroscopy was used to characterize the single tryptophan of GalR and to investigate the interaction between galactose and GalR. Fluorescence quenching experiments place both tryptophan residues of the GalR dimer in similar, solvent-exposed locations. Galactose is shown to enhance the intrinsic tryptophan fluorescence of GalR, the source of which is not explained by a change in decay times, but is due to an increase in the pre-exponential factor of the longest of the three fluorescence decay times. It is shown that the beta-anomer of D-galactose is the likely form that binds to GalR. An increase in pH from 6.3 to 9.5 causes the equilibrium association constant (K alpha) describing the galactose-GalR interaction to decrease 10-fold. The interaction is cooperative below pH 9.5. Over the pH range of 6.3 to 9.5, the tryptophan solvent exposure of GalR increases. Galactose binding also induces an increase in exposure. These results, and others presented in this paper, show that both pH and galactose cause global alterations in the structure of GalR.
Brown
M P
MP
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Shaikh
N
N
Brenowitz
M
M
Brand
L
L
D000494:Allosteric Regulation; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005690:Galactose / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011485:Protein Binding; D012097:Repressor Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050993:Galactose repressor proteins; D012097:Repressor Proteins; D005690:Galactose