Ligand‐Induced conformational changes in the lactose permease of escherichia coli: Evidence for two binding sites

Abstract

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331 + Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, 0,D-galactopyranosyl I-thio-0,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 33 1 + Cys permease was then purified and studied in dodecyl-0,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331 "+ Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (<0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331 + Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 --t Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%. Finally, the EPR spectrum of nitroxide spin-labeled Val 331 + Cys permease exhibits 2 components with different mobilities, and TDG causes the immobilized component to increase. The results provide evidence for the argument that lac permease has more than a single binding site. TDG binding to a higher affinity site quenches the fluorescence of MIANS-labeled Val 33 1 + Cys permease, and occupation of a second lower affinity site causes position 33 1 to become more accessible from a hydrophobic environment.Keywords: C-less permease; ligand-protein interaction; MIANS; site-directed fluorescence; site-directed spin labeling Lactose permease of Escherichia coli is a hydrophobic, polytopic, plasma membrane protein that catalyzes the coupled stoichiometric translocation of 0-galactosides and H + (reviewed in Kaback, , 1989Kaback, , 1992. The permease is encoded by the lacy Reprint requests to: H. Ronald Kaback, HHMI/UCLA, 6-720 MacDonald Building, 10833 Le Conte Avenue, Los Angeles, California 90024-1662; e-mail: ronaldk@hhmi.ucla.edu.Abbreviurions: lac, lactose; C-less permease, functional lac permease devoid of Cys residues; NEM, N-ethylmaleimide; CPM, 7-diethylamino-3-(4"maleimidylphenyl)-4-methylcoumarin; MIANS, 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid; A H + , the H + electrochemical gradient across the membrane; dodecylmaltoside, dodecyl-0,D-maltoside; KPi, potassium phosphate; TDG, 6,D-galctopyranosyl 1-thio-0,D-galactopyranoside;...

Fluorescence of native single‐Trp mutants in the lactose permease from Escherichia coli: Structural properties and evidence for a substrate‐induced conformational change

“…Scatchard analysis of TDG enhancement of MIANS-labeled W33C permease fluorescence reveals a single binding site with an apparent K O of 71 NM, which coincides with the high-affinity site described previously (Lolkema & Walz, 1990;Lolkema et al, 1991;van Iwaarden et al, 1993;Wu et al, 1995b). Therefore, it appears that occupancy of a high-affinity site in lactose permease causes position 33 to move into a more hydrophobic environment that is less accessible to MIANS, and positions 28 and 3 1 to move into a more hydrophilic environment that is more accessible to the probe.…”

“…The probe has been used extensively to study the reactivity of various single-Cys permease mutants Wu et al, 1995b). As shown in Figure 6, W33C permease reacts with MIANS, as evidenced by the linear increase in fluorescence for about 10 min after exposure to the probe.…”

Protonation and sugar binding to LacY

“…1). Numerous studies demonstrate that sugar binding at neutral pH induces a conformational change with LacY in the membrane (12,15) or with purified protein in detergent (13,14,28). Furthermore, rates of NPG binding measured by stoppedflow demonstrate that rapid sugar binding is followed by a slower conformational change detected with covalently bound MIANS as a reporter (31).…”

“…In the presence of galactopyranosides specifically, MIANS-labeled V331C LacY displays a decrease in fluorescence intensity (28,(30)(31)(32). Several other fluorophores were tested, and DACM-labeled V331C LacY was also found to be sensitive to sugar binding.…”

“…2A), and in this position, the fluorophore is sensitive to long-range conformational changes. This approach has been used to measure affinity for TDG (28), for detection of sugar binding to LacY mutants (29,30), and for stopped-flow kinetic studies (31). It is noteworthy that binding is measured indirectly, but the fluorescence changes observed are the immediate result of sugar binding.…”