Site-directed spin-labeling of transmembrane domain VII and the 4B1 antibody epitope in the lactose permease of Escherichia coli.
Functional lactose permease mutants containing single Cys residues at positions 233-255 and a biotin acceptor domain at the C terminus were solubilized in dodecyl beta-d-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol-selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy (EPR). The EPR spectral line shapes and the influence of nonpolar O2 or polar potassium chromium oxalate relaxation agents on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to the relaxation agents, respectively. The results provide evidence that residues Ser233-Asn246 are within the hydrophobic core of the membrane and that Phe247 is at the lipid headgroup-solvent interface. Along with Phe247, Phe250 and Gly254 are also surface-exposed, as indicated by studies on the epitope for monoclonal antibody 4B1 [Sun, J., Wu, J., Carasco, N., and Kaback, H. R. (1996) Biochemistry 35, 990-998]. Furthermore, the nitroxide-labeled intramembrane Cys replacements exhibit variations in mobility and accessibility that are consistent with the conclusion that TM VII is an alpha-helix in contact with surrounding helices in the tertiary structure of the permease.
Voss
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles, California 90095-1662, USA.
Hubbell
W L
WL
Hernandez-Borrell
J
J
Kaback
H R
HR
D000595:Amino Acid Sequence; D004578:Electron Spin Resonance Spectroscopy; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D013113:Spin Labels; D027981:Symporters
D000939:Epitopes; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013113:Spin Labels; D027981:Symporters; C021499:lactose permease