Helix packing in the lactose permease determined by metal-nitroxide interaction

John C Voss, Wayne L. Hubbell, H. Ronald Kaback

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances within proteins [Voss, J., Salwinski, L., Kaback, H. R., and Hubbell, W. L. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12295-12299; Voss, J., Hubbell, W. L., and Kaback, H. R. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 12300-12303], and the approach is utilized here to measure helix proximities in the lactose permease of Escherichia coli. A high-affinity divalent metal binding site was created by replacing Arg302 (helix IX) and Glu325 (helix X) with His residues in permease mutants containing single Cys residues in helices II, V, or VII and a biotin acceptor domain to facilitate purification. Mutant proteins were purified by avidin affinity chromatography, labeled specifically with a nitroxide free radical and investigated by electron paramagnetic resonance spectroscopy in the absence or presence of Cu(II). Spectral broadening due to bound Cu(II) was used to estimate distances between the metal center and the spin-labeled side chains. For each of the transmembrane domains probed, the variation in interspin distance with sequence position is consistent with an α-helical structure. The measured distances were also used to construct a model that is in good agreement with packing data obtained from other approaches.

Original languageEnglish (US)
Pages (from-to)211-216
Number of pages6
JournalBiochemistry
Volume37
Issue number1
DOIs
StatePublished - Jan 6 1998
Externally publishedYes

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Metals
Affinity chromatography
Membrane Transport Proteins
Avidin
Electron Spin Resonance Spectroscopy
Mutant Proteins
Biotin
Affinity Chromatography
Escherichia coli
Free Radicals
Purification
Paramagnetic resonance
Spectrum Analysis
Binding Sites
Spectroscopy
lactose permease
Proteins
nitroxyl

ASJC Scopus subject areas

  • Biochemistry

Cite this

Helix packing in the lactose permease determined by metal-nitroxide interaction. / Voss, John C; Hubbell, Wayne L.; Kaback, H. Ronald.

In: Biochemistry, Vol. 37, No. 1, 06.01.1998, p. 211-216.

Research output: Contribution to journalArticle

Voss, John C ; Hubbell, Wayne L. ; Kaback, H. Ronald. / Helix packing in the lactose permease determined by metal-nitroxide interaction. In: Biochemistry. 1998 ; Vol. 37, No. 1. pp. 211-216.
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