The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity

Tsai, I-Jung, Liu, Zhen-Wei, Rayment, John, Norman, Christel, McKinley, Allen and Martinac, Boris (2005) The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity. European Biophysics Journal, 34 5: 403-412. doi:10.1007/s00249-005-0476-x

Author Tsai, I-Jung
Liu, Zhen-Wei
Rayment, John
Norman, Christel
McKinley, Allen
Martinac, Boris
Title The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity
Journal name European Biophysics Journal   Check publisher's open access policy
ISSN 0175-7571
Publication date 2005-07-01
Sub-type Article (original research)
DOI 10.1007/s00249-005-0476-x
Volume 34
Issue 5
Start page 403
End page 412
Total pages 10
Place of publication Heidelberg, Germany
Publisher Springer
Language eng
Abstract The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively (Maurer and Dougherty J. Biol. Chem. 278(23):21076-21082, 2003). We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (124CSL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin-spin interaction with the neighboring 124C-SL residues in the MscL channel pentamer (Perozo et al.Nature 418:942-948, 2002). The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity.
Keyword Biophysics
mechanosensitive channel
electron paramagnetic resonance
Q-Index Code C1
Q-Index Status Provisional Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Excellence in Research Australia (ERA) - Collection
School of Biomedical Sciences Publications
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Created: Thu, 20 Sep 2007, 04:08:52 EST