>>Published online before print October 17, 2003
Proc. Natl. Acad. Sci. USA, 10.1073/pnas.1835645100
Biophysics
Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli
( bioenergetics | transport | fluorescence/phosphorescence | hydrophobic stacking | sugar binding )
José Luis Vázquez-Ibar *, Lan Guan *, Maja Svrakic, and H. Ronald Kaback
Howard Hughes Medical Institute and Departments of Physiology and Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662
Contributed by H. Ronald Kaback, September 3, 2003
The crystal structure of the Escherichia coli lactose permease at 3.5 Å 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.<<
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