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

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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 (I24C-SL) 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 I24C-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.

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Abbreviations

ANOVA:

Analysis of variance

D/R:

Dehydration/rehydration

EPR:

Electron paramagnetic resonance

GOF:

Gain of function

GST:

Glutathione S-transferase

Hepes:

N-(2-Hydroxyethyl)piperazine-N′-ethanesulfonic acid

ID:

Inner diameter

IPTG:

Isopropyl-β-D-thiogalactoside

LB:

Luria–Bertani

LOF:

Loss of function

LPC:

Lysophosphatidylcholine

MS:

Mechanosensitive

MscK:

Potassium-regulated mechanosensitive channel

MscL:

Mechanosensitive channel of large conductance

MscS:

Mechanosensitive channel of small conductance

OD:

Outer diameter

PBS:

Phosphate-buffered saline

SD:

Standard deviation

SDSL:

Site-directed spin labeling

TM1:

First transmembrane domain

TM2:

Second transmembrane domain

TPX:

Specially designed polymethylpentene

WT:

Wild type

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Acknowledgements

We thank Grischa Meyer for providing a ribbon diagram of MscL. This work was supported by the Australian Research Council.

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Author notes

  1. Boris Martinac

    Present address: School of Biomedical Sciences, University of Queensland, Brisbane, QLD, 4072, Australia

Authors and Affiliations

  1. School of Medicine and Pharmacology, University of Western Australia, QE II Medical Center, Nedlands, WA, 6009, Australia

    I-Jung Tsai, Zhen-Wei Liu, John Rayment, Christel Norman & Boris Martinac

  2. School of Biomedical and Chemical Sciences, University of Western Australian, 35 Stirling Highway, Crawley, WA, 6009, Australia

    I-Jung Tsai, John Rayment & Allan McKinley

  3. School of Biomedical Sciences, University of Queensland, Brisbane, QLD, 4072, Australia

    Zhen-Wei Liu

Authors
  1. I-Jung Tsai
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  2. Zhen-Wei Liu
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  6. Boris Martinac
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Correspondence to Boris Martinac.

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A Proceeding of the 28th Annual Meeting of the Australian Society for Biophysics

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Tsai, IJ., Liu, ZW., Rayment, J. et al. The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity. Eur Biophys J 34, 403–412 (2005). https://doi.org/10.1007/s00249-005-0476-x

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  • DOI: https://doi.org/10.1007/s00249-005-0476-x

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