Engineering a Metal Binding Site within a Polytopic Membrane Protein, the Lactose Permease of Escherichia coli

Abstract

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coli lie in close proximity [Jung, K., Jung, H., Wu, J., Privé, G. G., & Kaback, H. R. (1993) Biochemistry 32, 12273]. In this study, Glu269 was replaced with His in wildtype permease, leading to the presence of bis-His residues between helices VIII and X. Wild-type and Glu269→His permease containing a biotin acceptor domain were purified by monomeric avidin affinity chromatography, and binding of Mn2+ was studied by electron paramagnetic resonance (EPR) spectroscopy. The amplitude of the Mn2+ EPR spectrum is reduced by the Glu269→ His mutant, while no change is observed in the presence of wild-type permease. The Glu269→His mutant contains a single binding site for Mn2+ with a Kn of about 43 µM, and Mn2+ binding is pH dependent with no binding at pH 5.0, stoichiometric binding at pH 7.5, and a midpoint at about pH 6.3. The results confirm the conclusion that helices VIII and X are closely opposed in the tertiary structure of lac permease and provide a novel approach for studying helix proximity, as well as solvent accessibility, in polytopic membrane proteins. © 1995, American Chemical Society. All rights reserved.