title: The Effect of Detergent, Temperature, and Lipid on the Oligomeric State of MscL Constructs: Insights from Mass Spectrometry.
abstract: The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology.
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Eamonn
authors.initials: E
authors.lastname: Reading
authors.affiliation: Division of Chemistry and Chemical Engineering 114-96, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Troy A
authors.initials: TA
authors.lastname: Walton
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Idlir
authors.initials: I
authors.lastname: Liko
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Michael T
authors.initials: MT
authors.lastname: Marty
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3TA, UK.
authors.forename: Arthur
authors.initials: A
authors.lastname: Laganowsky
authors.affiliation: Division of Chemistry and Chemical Engineering 114-96, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Rees
authors.affiliation: Department of Chemistry, Physical & Theoretical Chemistry Laboratory, University of Oxford, Oxford OX1 3TA, UK. Electronic address: carol.robinson@chem.ox.ac.uk.
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008070:Lipopolysaccharides / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008055:Lipids; D008070:Lipopolysaccharides; C086596:MscL protein, E coli; D011993:Recombinant Fusion Proteins