The proximity between helix I and helix XI in the melibiose carrier of Escherichia coli as determined by cross-linking.
The melibiose carrier of Escherichia coli is a transmembrane protein that comprises 12 transmembrane helices connected by periplasmic and cytoplasmic loops, with both the N- and C-termini located on the cytoplasmic side. Our previous studies of second-site revertants suggested proximity between several helices, including helices XI and I. In this study, we constructed six double cysteine mutants, each having one cysteine in helix I and the other in helix XI: three mutants, K18C/S380C, D19C/S380C, and F20C/S380C, have their cysteine pairs near the cytoplasmic side of the carrier, and the other three, T34C/G395C, D35C/G395C, and V36C/G395C, have their cysteine pairs near the periplasmic side. In the absence of substrate, disulfide formations catalyzed by iodine and copper-(1,10-phenanthroline)(3) indicate that helix I and helix XI are in immediate proximity to each other on the periplasmic side but not on the cytoplasmic side, as shown by protease cleavage analyses. We infer that the two helices are tilted with respect to each other, with the periplasmic sides in close proximity.
Ding
P Z
PZ
Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Wilson
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TH
D000595:Amino Acid Sequence; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics*; D018257:Helix-Loop-Helix Motifs; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D027981:Symporters
D003432:Cross-Linking Reagents; D026901:Membrane Transport Proteins; D027981:Symporters; C021548:melibiose permease; D003545:Cysteine