Molecular Biology and Energetics of Active Transport

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

“…In this report, we document studies on V331C permease in membrane vesicles and after solubilization and purification in dodecylmaltoside. V331 is located close to the cytoplasmic surface of helix X, a domain containing His 322 and Glu 325, which are essential for active lactose transport (see Kaback, 1989Kaback, , 1992, and Lys 319, which interacts via a putative salt bridge with Asp 240 in helix VI1 but is not essential for activity (see Kaback et al, 1993). On the other hand, as demonstrated by Cys-scanning mutagenesis (SahinToth & , each of the other residues in helix X can be replaced with Cys with retention of highly significant transport activity.…”

“…Based on CD and the hydropathy analysis of the primary amino acid sequence, a secondary-structure model was proposed (Foster et al, 1983) in which the permease is composed of 12 hydrophobic segments in a-helical conformation that traverse the membrane in zig-zag fashion connected by hydrophilic domains (loops) with both the N-and C-termini on the cytoplasmic face. Evidence favoring general aspects of the model and showing that the C-terminus, as well as the second and third cytoplasmic loops, are on the cytoplasmic face of the membrane has been obtained from a variety of experimental approaches (see Kaback, , 1989Kaback, , 1992. Moreover, analysis of a large number of lac permease-alkaline phosphatase (lacy-phoA) fusions has provided unequivocal support for the topological predictions of the 12-helices model (Calamia & Manoil, 1990).…”

“…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.…”

“…Site-directed mutagenesis has allowed delineation of certain amino acid residues in lac permease that are essential for the transport activity and/or substrate binding (Kaback, 1989(Kaback, , 1992Kaback et al, 1993). However, it has become clear that structural and dynamic information at high resolution is required to understand the role of these residues in the transport mechanism .…”

“…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.…”