PMID:15812636
| Citation |
Tsai, IJ, Liu, ZW, Rayment, J, Norman, C, McKinley, A and Martinac, B (2005) The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity. Eur. Biophys. J. 34:403-12 |
|---|---|
| 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. 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. 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. |
| Links |
PubMed Online version:10.1007/s00249-005-0476-x |
| Keywords |
Arginine/chemistry; Cysteine/chemistry; DNA Mutational Analysis; Electron Spin Resonance Spectroscopy; Electrophysiology; Escherichia coli/metabolism; Escherichia coli Proteins/chemistry; Genes, Reporter; Glutamine/chemistry; Glutathione Transferase/metabolism; Ion Channels/chemistry; Isoleucine/chemistry; Leucine/chemistry; Lipid Bilayers/chemistry; Lipids/chemistry; Liposomes/chemistry; Lysophosphatidylcholines/chemistry; Models, Biological; Models, Molecular; Mutagenesis, Site-Directed; Mutation; Patch-Clamp Techniques; Periplasm/metabolism; Phenotype; Pressure; Protein Conformation; Protein Structure, Tertiary; Spin Labels; Temperature |
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