Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an α-helix

John C Voss, Molly M. He, Wayne L. Hubbell, H. Ronald Kaback

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

Functional lactose permease mutants containing single-Cys residues at positions 387-402 [He, M. M., Sun, J., and Kaback, H. R. (1996) Biochemistry 35, 12909-12914] and a biotin acceptor domain in the middle cytoplasmic loop were solubilized in n-dodecyl-β-D-maltopyranoside and purified by avidin affinity chromatography. Each mutant protein was derivatized with a thiol- selective nitroxide reagent and examined by conventional and power saturation electron paramagnetic resonance spectroscopy. Analysis of the electron paramagnetic resonance spectral line shapes and the influence of O2 on the saturation behavior of the spin-labeled proteins were measured in order to obtain information on the mobility of the spin-labeled side chains and their accessibility to O2, respectively. The data show a periodic dependence of both mobility and accessibility on sequence position consistent with an α- helical structure. These results provide direct support for the contention that transmembrane domain XII is in an α-helical conformation and on the periphery of the 12-helix bundle that comprises the lactose permease molecule.

Original languageEnglish (US)
Pages (from-to)12915-12918
Number of pages4
JournalBiochemistry
Volume35
Issue number39
DOIs
StatePublished - 1996

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Electron Spin Resonance Spectroscopy
Labeling
Escherichia coli
Paramagnetic resonance
Affinity chromatography
Biochemistry
Avidin
Mutant Proteins
Biotin
Affinity Chromatography
Sulfhydryl Compounds
Conformations
Spectrum Analysis
Spectroscopy
Molecules
Proteins
lactose permease
dodecyl maltopyranoside

ASJC Scopus subject areas

  • Biochemistry

Cite this

Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an α-helix. / Voss, John C; He, Molly M.; Hubbell, Wayne L.; Ronald Kaback, H.

In: Biochemistry, Vol. 35, No. 39, 1996, p. 12915-12918.

Research output: Contribution to journalArticle

Voss, John C ; He, Molly M. ; Hubbell, Wayne L. ; Ronald Kaback, H. / Site-directed spin labeling demonstrates that transmembrane domain XII in the lactose permease of Escherichia coli is an α-helix. In: Biochemistry. 1996 ; Vol. 35, No. 39. pp. 12915-12918.
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