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 journalArticlepeer-review

39 Scopus citations

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

ASJC Scopus subject areas

  • Biochemistry

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