EFFECT OF LIPID PACKING ON THE CONFORMATIONAL STATES OF PURIFIED GLUT-1 HEXOSE TRANSPORTER

The purpose of this study was to determine the effect of increased Lip id packing on the conformational states of the GLUT-1 hexose transport er purified in endogenous lipids. The binding of glucose results in a conformational change that can be followed by a decrease in fluorescen ce intensity. Lipid packing was increased by subjecting the samples to hydrostatic pressure. We have found that in the absence of ligand, th e fluorescence intensity decreased approximately 20% in the 600 bar ra nge studied. In the presence of either saturating or half-saturating a mounts of D-glucose, a substantial loss in intensity (approximately 80 %) was observed. Similar decreases were also seen the presence of a gl ucose analog, maltose, or a noncompetitive inhibitor, cytochalasin B. Changes in the accessibility of aqueous soluble quenchers (I- and acry lamide) to GLUT-1 Trp and Tyr residues suggested that ligand binding c auses interfacial fluorophores to move closer to ionic groups in the l ipid head group region of the membrane. This idea was substantiated by (1) increased static quenching of the GLUT-1 fluorophores in the pres ence of ligand, (2) increased energy transfer efficiency between GLUT- 1 fluorophores and a fluorescent membrane probe located close to the h ead group region, and (3) reduced change in rotational motion with tem perature in the presence of ligand. Since the application of pressure results in an increase in bilayer thickness, and ligand binding causes a population of fluorophores to move closer to the membrane surface, then these interfacial interactions can be more stabilized under press ure. Studies monitoring the change in quenching of membrane probes by GLUT-1 tryptophans and energy transfer of GLUT-1 tryptophans to membra ne probes support this idea.