title: Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes.
abstract: In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.|School of Chemistry, Monash University, Melbourne 3800, Australia.|Department of Chemical Engineering, Monash University, Melbourne 3800.
authors.forename: Hsin-Hui
authors.initials: HH
authors.lastname: Shen
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Denisse L
authors.initials: DL
authors.lastname: Leyton
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Takuya
authors.initials: T
authors.lastname: Shiota
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Belousoff
authors.affiliation: NIDDK, NIH, Bethesda 20892, United States of America.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Noinaj
authors.affiliation: Department of Chemical Engineering, Monash University, Melbourne 3800.
authors.forename: Jingxiong
authors.initials: J
authors.lastname: Lu
authors.affiliation: The Bragg Institute, Australian Nuclear Science and Technology Organization (ANSTO), Lucas Heights 2234, Australia.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Holt
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Khershing
authors.initials: K
authors.lastname: Tan
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Joel
authors.initials: J
authors.lastname: Selkrig
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Chaille T
authors.initials: CT
authors.lastname: Webb
authors.affiliation: NIDDK, NIH, Bethesda 20892, United States of America.
authors.forename: Susan K
authors.initials: SK
authors.lastname: Buchanan
authors.affiliation: School of Chemistry, Monash University, Melbourne 3800, Australia.
authors.forename: Lisandra L
authors.initials: LL
authors.lastname: Martin
authors.affiliation: Department of Microbiology, Monash University, Melbourne 3800, Australia.
authors.forename: Trevor
authors.initials: T
authors.lastname: Lithgow
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000378:metabolism; D008958:Models, Molecular; D016254:Mutagenesis, Insertional / Q000235:genetics; D053758:Nanoparticles / Q000737:chemistry*; D017433:Protein Structure, Secondary; D058525:Quartz Crystal Microbalance Techniques; D013379:Substrate Specificity
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D008051:Lipid Bilayers