The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity.
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.
Tsai
I-Jung
IJ
School of Medicine and Pharmacology, University of Western Australia, QE II Medical Center, Nedlands, WA 6009, Australia.
Liu
Zhen-Wei
ZW
Rayment
John
J
Norman
Christel
C
McKinley
Allan
A
Martinac
Boris
B
D001120:Arginine / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004252:DNA Mutational Analysis; D004578:Electron Spin Resonance Spectroscopy; D004594:Electrophysiology; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry*; D017930:Genes, Reporter; D005973:Glutamine / Q000737:chemistry*; D005982:Glutathione Transferase / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry*; D007532:Isoleucine / Q000737:chemistry; D007930:Leucine / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008055:Lipids / Q000737:chemistry; D008081:Liposomes / Q000737:chemistry; D008244:Lysophosphatidylcholines / Q000737:chemistry; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D018408:Patch-Clamp Techniques; D019897:Periplasm / Q000378:metabolism*; D010641:Phenotype; D011312:Pressure; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013113:Spin Labels; D013696:Temperature
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008055:Lipids; D008081:Liposomes; D008244:Lysophosphatidylcholines; C086596:MscL protein, E coli; D013113:Spin Labels; D007532:Isoleucine; D005973:Glutamine; D001120:Arginine; D005982:Glutathione Transferase; D007930:Leucine; D003545:Cysteine