Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

Qingda Wang; John Voss; Wayne L. Hubbell; H. Ronald Kaback

doi:10.1021/bi972990f

Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

Qingda Wang, John Voss, Wayne L. Hubbell, H. Ronald Kaback

Research output: Contribution to journal › Article › peer-review

19 Scopus citations

Abstract

Three double-Cys mutant pairs - Ala→273Cys/Met299→Cys, Thr266→Cys/Ile303→Cys, and Thr266→Cys/Ser306→Cys - were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273→Cys/Met299→Cys, but not with the single-Cys mutants nor with mutants Thr266→Cys/Ile303→Cys or Thr266→Cys/Ser306→Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273→Cys/Met299→Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

Original language	English (US)
Pages (from-to)	4910-4915
Number of pages	6
Journal	Biochemistry
Volume	37
Issue number	14
DOIs	https://doi.org/10.1021/bi972990f
State	Published - Apr 7 1998
Externally published	Yes

ASJC Scopus subject areas

Biochemistry

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@article{a0bbf93d87994d768ca1ea8d15e4bd03,

title = "Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli",

abstract = "Three double-Cys mutant pairs - Ala→273Cys/Met299→Cys, Thr266→Cys/Ile303→Cys, and Thr266→Cys/Ser306→Cys - were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273→Cys/Met299→Cys, but not with the single-Cys mutants nor with mutants Thr266→Cys/Ile303→Cys or Thr266→Cys/Ser306→Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273→Cys/Met299→Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.",

author = "Qingda Wang and John Voss and Hubbell, {Wayne L.} and Kaback, {H. Ronald}",

year = "1998",

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T1 - Proximity of helices VIII (Ala273) and IX (Met299) in the lactose permease of Escherichia coli

AU - Wang, Qingda

AU - Voss, John

AU - Hubbell, Wayne L.

AU - Kaback, H. Ronald

PY - 1998/4/7

Y1 - 1998/4/7

N2 - Three double-Cys mutant pairs - Ala→273Cys/Met299→Cys, Thr266→Cys/Ile303→Cys, and Thr266→Cys/Ser306→Cys - were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273→Cys/Met299→Cys, but not with the single-Cys mutants nor with mutants Thr266→Cys/Ile303→Cys or Thr266→Cys/Ser306→Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273→Cys/Met299→Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

AB - Three double-Cys mutant pairs - Ala→273Cys/Met299→Cys, Thr266→Cys/Ile303→Cys, and Thr266→Cys/Ser306→Cys - were constructed in a functional lac permease construct devoid of Cys residues, and the excimer fluorescence or electron paramagnetic resonance (EPR) was studied with pyrene- or spin-labeled derivatives, respectively. After reconstitution into proteoliposomes, excimer fluorescence is observed with mutant Ala273→Cys/Met299→Cys, but not with the single-Cys mutants nor with mutants Thr266→Cys/Ile303→Cys or Thr266→Cys/Ser306→Cys. Furthermore, spin-spin interaction is also observed with mutant Ala273→Cys/Met299→Cys, but only after the permease is reconstituted into proteoliposomes. The results provide independent support for the conclusions that helix VIII is close to helix IX and that the transmembrane helices of the permease are more loosely packed in a detergent micelle as opposed to a phospholipid bilayer.

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