Exploiting luminescence spectroscopy to elucidate the interaction between sugar and a tryptophan residue in the lactose permease of Escherichia coli

AUTOR(ES)

Vázquez-Ibar, José Luis

FONTE

National Academy of Sciences

RESUMO

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.

Documentos Relacionados

• Functional interactions between putative intramembrane charged residues in the lactose permease of Escherichia coli.
• The substrate-binding site in the lactose permease of Escherichia coli
• Unraveling the mechanism of the lactose permease of Escherichia coli
• The mechanism of inducer exclusion. Direct interaction between purified IIIGlc of the phosphoenolpyruvate:sugar phosphotransferase system and the lactose carrier of Escherichia coli
• Transport by the lactose permease of Escherichia coli as the basis of lactose killing.