Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an alpha-helix.
Functional lactose permease mutants containing single-Cys residues at positions 387-402 [He, M. M., Sun, J., & Kaback, H. R. (1996) Biochemistry 35, 12909-12914] and a biotin acceptor domain in the middle cytoplasmic loop were solubilized in n-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. Analysis of the electron paramagnetic resonance spectral line shapes and the influence of O2 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 O2, respectively. The data show a periodic dependence of both mobility and accessibility on sequence position consistent with an alpha-helical structure. These results provide direct support for the contention that transmembrane domain XII is in an alpha-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permease molecule.
Voss
J
J
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
He
M M
MM
Hubbell
W L
WL
Kaback
H R
HR
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009004:Monosaccharide Transport Proteins; D009154:Mutation; D010100:Oxygen; D017433:Protein Structure, Secondary; D013113:Spin Labels; D027981:Symporters
D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D013113:Spin Labels; D027981:Symporters; C021499:lactose permease; D010100:Oxygen