Ligand-dependent quenching of tryptophan fluorescence in human erythrocyte hexose transport protein.
D-Glucose transport by the 492-residue human erythrocyte hexose transport protein may involve ligand-mediated conformational/positional changes. To examine this possibility, hydrophilic quencher molecules [potassium iodide and acrylamide (ACR)] were used to monitor the quenching of the total protein intrinsic fluorescence exhibited by the six protein tryptophan (Trp) residues in the presence and absence of substrate D-glucose, and in the presence of the inhibitors maltose and cytochalasin B. Protein fluorescence was found to be quenched under various conditions, ca. 14-24% by KI and ca. 25-33% by ACR, indicating that the bulk of the Trp residue population occurs in normally inaccessible hydrophobic regions of the erythrocyte membrane. However, in the presence of D-glucose, quenching by KI and ACR decreased an average of -3.4% and -4.4%, respectively; Stern-Volmer plots displayed decreased slopes in the presence of D-glucose, confirming the relatively reduced quenching. In contrast, quenching efficiency increased in the presence of maltose (+5.9%, +3.3%), while addition of cytochalasin B had no effect on fluorescence quenching. The overall results are interpreted in terms of ligand-activated movement of an initially aqueous-located protein segment containing a Trp residue into, or toward, the cellular membrane. Relocation of this segment, in effect, opens the D-glucose channel; maltose and cytochalasin B would thus inhibit transport by mechanisms which block this positional change. Conformational and hydropathy analyses suggested that the region surrounding Trp-388 is an optimal "dynamic segment" which, in response to ligand activation, could undergo the experimentally deduced aqueous/membrane domain transfer.
Pawagi
A B
AB
Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Deber
C M
CM
D000178:Acrylamides / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D004912:Erythrocytes / Q000378:metabolism*; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D006801:Humans; D008024:Ligands; D008320:Maltose / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000097:blood*; D011193:Potassium Iodide / Q000494:pharmacology; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
D000178:Acrylamides; D008024:Ligands; D009004:Monosaccharide Transport Proteins; D011193:Potassium Iodide; D008320:Maltose; D014364:Tryptophan; D005947:Glucose