Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli.
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 wild-type 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 KD of about 43 microM, 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.
Jung
K
K
Howard Hughes Medical Institute, Department of Physiology, University of California, Los Angeles 90095-1662, USA.
Voss
J
J
He
M
M
Hubbell
W L
WL
Kaback
H R
HR
D001665:Binding Sites; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005971:Glutamates; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D008345:Manganese / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D008670:Metals; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D027981:Symporters
D029968:Escherichia coli Proteins; D005971:Glutamates; C050221:LacY protein, E coli; D026901:Membrane Transport Proteins; D008670:Metals; D009004:Monosaccharide Transport Proteins; D027981:Symporters; D008345:Manganese; D006639:Histidine; C021499:lactose permease