PROXIMITY OF HELICE-VIII (ALA273) AND HELICE-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 func tional 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 reconstitutio n into proteoliposomes, excimer fluorescence is observed with mutant A la273-->Cys/Met299-->Cys, but not with the single-Cys mutants nor with mutants Thr266-->Cys/Ile303-->Cys or Thr266-->Cys/Ser306-->Cys. Furth ermore, spin-spin interaction is also observed with mutant Ala273-->Cy s/Met299-->Cys, but only after the permease is reconstituted into prot eoliposomes. The results provide independent support for the conclusio ns that helix VIII is close to helix IX and that the transmembrane hel ices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.