title: OCAM: a new tool for studying the oligomeric diversity of MscL channels.
abstract: We have developed a new technique to study the oligomeric state of proteins in solution. OCAM or Oligomer Characterization by Addition of Mass counts protein subunits by selectively shaving a protein mass tag added to a protein subunit via a short peptide linker. Cleavage of each mass tag reduces the total mass of the protein complex by a fixed amount. By performing limited proteolysis and separating the reaction products by size on a blue native PAGE gel, a ladder of reaction products corresponding to the number of subunits can be resolved. The pattern of bands may be used to distinguish the presence of a single homo-oligomer from a mixture of oligomeric states. We have applied OCAM to study the mechanosensitive channel of large conductance (MscL) and find that these proteins can exist in multiple oligomeric states ranging from tetramers up to possible hexamers. Our results demonstrate the existence of oligomeric forms of MscL not yet observed by X-ray crystallography or other techniques and that in some cases a single type of MscL subunit can assemble as a mixture of oligomeric states.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
authors.forename: Chris S
authors.initials: CS
authors.lastname: Gandhi
authors.forename: Troy A
authors.initials: TA
authors.lastname: Walton
authors.forename: Douglas C
authors.initials: DC
authors.lastname: Rees
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007473:Ion Channels / Q000737:chemistry*; D016014:Linear Models; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry*; D016415:Sequence Alignment
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; C086596:MscL protein, E coli; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins