Proximity between Glu126 and Arg144 in the lactose permease of Escherichiacoli

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 ( helix V) which are critical for substrate binding in the lactose permease o f Escherichia coli are charge paired and therefore in close proximity. To t est this conclusion more directly, three different site-directed spectrosco pic techniques were applied to permease mutants in which Glu126 and/or Arg1 44 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-- >His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electro n paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 mu M at pH 7.5, while no binding is observed at pH 5.5. I n addition, no binding is detected with Glu126-->His or Argl44-->His permea se. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domai n was purified, labeled with a thiol-specific nitroxide spin-label, and sho wn to exhibit spin-spin interactions in the frozen state after reconstituti on into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biot in acceptor domain was purified and labeled with a thiol-specific pyrene de rivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R1 44C mutant, but not with either single-Cys mutant or when the single-Cys mu tants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in clos e physical proximity.