Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

Biochemistry
Q WangH R Kaback

Abstract

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.

Citations

Nov 26, 1998·Hepatology : Official Journal of the American Association for the Study of Liver Diseases·A W WolkoffB F Scharschmidt
Mar 30, 2002·Advanced Drug Delivery Reviews·Eric Y ZhangPeter W Swaan
Nov 18, 1998·Current Opinion in Structural Biology·W L HubbellM A Lietzow
Oct 23, 2002·Proceedings of the National Academy of Sciences of the United States of America·Paul L SorgenMark E Girvin
Oct 8, 1998·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·S FrillingosH R Kaback
Jan 5, 2002·Journal of Molecular Biology·Roman A MelnykCharles M Deber

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