S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro

Implications for detergent- solubilized membrane proteins

Michael R. Dorwart, Robin Wray, Chad A. Brautigam, Youxing Jiang, Paul Blount

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C8E5 detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.

Original languageEnglish (US)
Article numbere1000555
JournalPLoS Biology
Volume8
Issue number12
DOIs
StatePublished - Dec 2010

Fingerprint

stoichiometry
membrane proteins
Stoichiometry
detergents
Detergents
Membrane Proteins
X ray crystallography
X Ray Crystallography
X-ray diffraction
Oligomerization
Centrifugation
Protein S
Octoxynol
light scattering
Cell membranes
crosslinking
sulfides
Sedimentation
tuberculosis
Disulfides

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)

Cite this

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title = "S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro: Implications for detergent- solubilized membrane proteins",
abstract = "While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C8E5 detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.",
author = "Dorwart, {Michael R.} and Robin Wray and Brautigam, {Chad A.} and Youxing Jiang and Paul Blount",
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T1 - S. aureus MscL is a pentamer in vivo but of variable stoichiometries in vitro

T2 - Implications for detergent- solubilized membrane proteins

AU - Dorwart, Michael R.

AU - Wray, Robin

AU - Brautigam, Chad A.

AU - Jiang, Youxing

AU - Blount, Paul

PY - 2010/12

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