Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli.
The crystal structure of the Escherichia coli lactose permease at 3.5 A with a bound substrate has been reported recently. The structure reveals the sugar-protein contacts, which include hydrophobic stacking between the galactopyranosyl ring of substrate and the indole side chain of Trp-151, as proposed previously. The nature of this interaction is studied here by exploiting the luminescence properties of Trp-151 in a mutant devoid of other tryptophan residues. The following phenomena are observed. (i) The fluorescence emission spectrum of Trp-151 and fluorescence-quenching experiments with water-soluble quenchers demonstrate that Trp-151 is in a hydrophilic environment. (ii) Substrate binding leads to a blue shift in the emission spectrum and reduction in accessibility to polar quenchers, indicating that Trp-151 becomes less exposed to aqueous solvent. (iii) The phosphorescence spectrum of Trp-151 is red-shifted in the presence of substrate, indicating charge separation of the triplet state due to a direct stacking interaction between the galactopyranosyl and indole rings. The spectroscopic data fully complement the x-ray structure and demonstrate the feasibility of fluorescence spectroscopy for studying sugar-protein interactions.
Vázquez-Ibar
José Luis
JL
Howard Hughes Medical Institute, and Department of Physiology, Molecular Biology Institute, University of California-Los Angeles, Los Angeles, CA 90095-1662, USA.
Guan
Lan
L
Svrakic
Maja
M
Kaback
H Ronald
HR
D019943:Amino Acid Substitution; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008163:Luminescent Measurements; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D027981:Symporters; D014364:Tryptophan / Q000378:metabolism*
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D014364:Tryptophan; C021499:lactose permease