Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli.
Three double-Cys mutant pairs--Ala273-->Cys/Met299-->Cys, Thr266-->Cys/Ile303-->Cys, and Thr266-->Cys/Ser306-->Cys--were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273-->Cys/Met299-->Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.
Wang
Q
Q
Howard Hughes Medical Institute, Department of Physiology, University of California Los Angeles, Los Angeles, California 90095-1662, USA.
Voss
J
J
Hubbell
W L
WL
Kaback
H R
HR
D000409:Alanine / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics; D008715:Methionine / Q000737:chemistry*; D009004:Monosaccharide Transport Proteins; D016297:Mutagenesis, Site-Directed; 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; D008715:Methionine; D000409:Alanine