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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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