PMID:14566061
| Citation |
Vázquez-Ibar, JL, Guan, L, Svrakic, M and Kaback, HR (2003) Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 100:12706-11 |
|---|---|
| Abstract |
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. |
| Links |
PubMed PMC240682 Online version:10.1073/pnas.1835645100 |
| Keywords |
Amino Acid Substitution; Binding Sites; Crystallography, X-Ray; Escherichia coli/enzymology; Escherichia coli Proteins; Kinetics; Luminescent Measurements; Membrane Transport Proteins/chemistry; Membrane Transport Proteins/metabolism; Monosaccharide Transport Proteins; Mutagenesis, Site-Directed; Protein Structure, Secondary; Spectrometry, Fluorescence; Symporters; Tryptophan/metabolism |
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