TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution

Markus Safferling, Heather Griffith, Jie Jin, Josh Sharp, Magdia De Jesus, Caroline L Ng, Terry A. Krulwich, Da Neng Wang

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.

Original languageEnglish (US)
Pages (from-to)13969-13976
Number of pages8
JournalBiochemistry
Volume42
Issue number47
DOIs
StatePublished - Dec 2 2003

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Bacilli
Tetracycline
Bacillus subtilis
Detergents
Dimers
Membranes
Proteins
Antiporters
Proteolysis
Divalent Cations
Post Translational Protein Processing
Size exclusion chromatography
Gel Chromatography
Protons
Polyacrylamide Gel Electrophoresis
Mass Spectrometry
Membrane Proteins
Oligomers
Escherichia coli
Restoration

ASJC Scopus subject areas

  • Biochemistry

Cite this

Safferling, M., Griffith, H., Jin, J., Sharp, J., De Jesus, M., Ng, C. L., ... Wang, D. N. (2003). TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution. Biochemistry, 42(47), 13969-13976. https://doi.org/10.1021/bi035173q

TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution. / Safferling, Markus; Griffith, Heather; Jin, Jie; Sharp, Josh; De Jesus, Magdia; Ng, Caroline L; Krulwich, Terry A.; Wang, Da Neng.

In: Biochemistry, Vol. 42, No. 47, 02.12.2003, p. 13969-13976.

Research output: Contribution to journalArticle

Safferling, M, Griffith, H, Jin, J, Sharp, J, De Jesus, M, Ng, CL, Krulwich, TA & Wang, DN 2003, 'TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution', Biochemistry, vol. 42, no. 47, pp. 13969-13976. https://doi.org/10.1021/bi035173q
Safferling, Markus ; Griffith, Heather ; Jin, Jie ; Sharp, Josh ; De Jesus, Magdia ; Ng, Caroline L ; Krulwich, Terry A. ; Wang, Da Neng. / TetL Tetracycline Efflux Protein from Bacillus subtilis Is a Dimer in the Membrane and in Detergent Solution. In: Biochemistry. 2003 ; Vol. 42, No. 47. pp. 13969-13976.
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N2 - The TetL antiporter from the Bacillus subtilis inner membrane is a tetracycline-divalent cation efflux protein that is energized by the electrochemical proton gradient across the membrane. In this study, we expressed tetL in Escherichia coli and investigated the oligomeric state of TetL in the membrane and in detergent solution. Evidence for an oligomeric state of TetL emerged from SDS-PAGE and Western blot analysis of membrane samples as well as purified protein samples from cells that expressed two differently tagged TetL species. Furthermore, no formation or restoration of TetL oligomers occurred upon detergent solubilization of the membrane. Rather, oligomeric forms established in vivo persisted after solubilization. Mass spectrometry of the purified protein showed the absence of proteolysis and posttranslational modifications. Analytical size-exclusion chromatography of the purified protein revealed a dimeric TetL in dodecyl-maltoside solution. In addition, TetL dimers were found in a number of other detergents and over a wide pH range. It is therefore likely that the oligomeric form of the protein in the membrane is also a dimer.

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