Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli

Kirsten Jung, John Voss, Molly He, John C Voss, H. Ronald Kaback

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Abstract

Site-directed excimer fluorescence indicates that Glu269 (helix VIII) and His322 (helix X) in the lactose permease of Escherichia coll 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 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.

Original languageEnglish (US)
Pages (from-to)6272-6277
Number of pages6
JournalBiochemistry®
Volume34
Issue number19
StatePublished - 1995

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Membrane Transport Proteins
Escherichia coli
Membrane Proteins
Metals
Binding Sites
Paramagnetic resonance
Electron Spin Resonance Spectroscopy
Affinity chromatography
Biochemistry
Avidin
Biotin
Escherichia
Fluorescence
Affinity Chromatography
Spectroscopy
Spectrum Analysis
lactose permease

ASJC Scopus subject areas

  • Biochemistry

Cite this

Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli. / Jung, Kirsten; Voss, John; He, Molly; Voss, John C; Kaback, H. Ronald.

In: Biochemistry®, Vol. 34, No. 19, 1995, p. 6272-6277.

Research output: Contribution to journalArticle

Jung, Kirsten ; Voss, John ; He, Molly ; Voss, John C ; Kaback, H. Ronald. / Engineering a metal binding site within a polytopic membrane protein, the lactose permease of Escherichia coli. In: Biochemistry®. 1995 ; Vol. 34, No. 19. pp. 6272-6277.
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