Use of site-directed fluorescence labeling to study proximity relationships in the lactose permease of Escherichia coli.
The lactose permease of Escherichia coli is a paradigm for polytopic membrane transport proteins that transduce free energy stored in an electrochemical ion gradient into work in the form of a concentration gradient. Although the permease consists of 12 hydrophobic transmembrane domains in probable alpha-helical conformation that traverse the membrane in zigzag fashion connected by hydrophilic "loops", little information is available regarding the folded tertiary structure of the molecule. In this paper, we describe an approach to studying proximity relationships in lactose permease that is based upon site-directed pyrene labeling of combinations of paired Cys replacements in a mutant devoid of Cys residues. Since pyrene exhibits excimer fluorescence if two molecules are within about 3.5 A, the proximity between paired labeled residues can be determined. The results demonstrate that putative helices VIII and IX are close to helix X. Taken together with other findings indicating that helix VII is close to helices X and XI, the data lead to a model that describes the packing of helices VII-XI.
Jung
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Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90024-1662.
Jung
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Wu
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Privé
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Kaback
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D001483:Base Sequence; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D027981:Symporters
D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; C021499:lactose permease