Surface-exposed positions in the transmembrane helices of the lactose permease of Escherichia coli determined by intermolecular thiol cross-linking.
Intermolecular thiol cross-linking was used to determine surface-exposed positions in 250 lactose permease mutants containing single-Cys replacements in each transmembrane helix. Significant cross-linking of monomers to produce homodimers is observed in nine mutants with a 5-A-long cross-linking agent containing bis-methane thiosulfonate reactive groups [position 78 (helix III); positions 185, 186, and 187 (helix VI); positions 263, 275, and 278 (helix VIII); and positions 308 (helix IX) and 398 (helix XII)]. The results are consistent with a current helix-packing model of the permease. Seven of the nine mutants that exhibit intermolecular cross-linking are located at or near the cytoplasmic ends of transmembrane helices; two are near periplasmic ends. The results suggest that only those Cys replacements accessible from the aqueous phase and not from the hydrophobic core of the membrane are susceptible to cross-linking because of the much higher reactivity of the thiolate anion relative to the thiol. Single-Cys mutants at positions 278 (helix VIII) and 398 (helix XII), which are located in opposite sides of the 12-helix bundle, exhibit similar rates of cross-linking with sigmoid kinetics. Furthermore, cross-linking is markedly decreased at 0 degrees C, suggesting that lateral diffusion of the permease within the plane of the membrane is important for intermolecular cross-linking. The findings confirm previous observations indicating that intermolecular cross-linking is a stochastic process resulting from random collisions and support a number of other lines of evidence that lactose permease is a monomer.
Guan
Lan
L
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California, Los Angeles, CA 90095-1662, USA.
Murphy
Franklin D
FD
Kaback
H Ronald
HR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003432:Cross-Linking Reagents / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004058:Diffusion; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008024:Ligands; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D017433:Protein Structure, Secondary; D013269:Stochastic Processes; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D027981:Symporters
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013438:Sulfhydryl Compounds; D027981:Symporters; C021499:lactose permease; D003545:Cysteine