title: Prepore Stability Controls Productive Folding of the BAM-independent Multimeric Outer Membrane Secretin PulD.
abstract: Members of a group of multimeric secretion pores that assemble independently of any known membrane-embedded insertase in Gram-negative bacteria fold into a prepore before membrane-insertion occurs. The mechanisms and the energetics that drive the folding of these proteins are poorly understood. Here, equilibrium unfolding and hydrogen/deuterium exchange monitored by mass spectrometry indicated that a loss of 4-5 kJ/mol/protomer in the N3domain that is peripheral to the membrane-spanning C domain in the dodecameric secretin PulD, the founding member of this class, prevents pore formation by destabilizing the prepore into a poorly structured dodecamer as visualized by electron microscopy. Formation of native PulD-multimers by mixing protomers that differ in N3domain stability, suggested that the N3domain forms a thermodynamic seal onto the prepore. This highlights the role of modest free energy changes in the folding of pre-integration forms of a hyperstable outer membrane complex and reveals a key driving force for assembly independently of the β-barrel assembly machinery.
authors.affiliation: From the Molecular Genetics Unit, CNRS ERL 3526.|Laboratory of Macromolecular Systems and Signaling and.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Guilvout
authors.affiliation: Structural Mass Spectrometry and Proteomics Unit, CNRS UMR 3528, Institut Pasteur, 75724 Paris Cedex 15, France and.
authors.forename: Sébastien
authors.initials: S
authors.lastname: Brier
authors.affiliation: the BioEM lab, Biozentrum, University of Basel, CH 4058 Basel, Switzerland.
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chami
authors.affiliation: Structural Mass Spectrometry and Proteomics Unit, CNRS UMR 3528, Institut Pasteur, 75724 Paris Cedex 15, France and.
authors.forename: Véronique
authors.initials: V
authors.lastname: Hourdel
authors.affiliation: From the Molecular Genetics Unit, CNRS ERL 3526.|Laboratory of Macromolecular Systems and Signaling and.
authors.forename: Olivera
authors.initials: O
authors.lastname: Francetic
authors.affiliation: From the Molecular Genetics Unit, CNRS ERL 3526.
authors.forename: Anthony P
authors.initials: AP
authors.lastname: Pugsley
authors.affiliation: Structural Mass Spectrometry and Proteomics Unit, CNRS UMR 3528, Institut Pasteur, 75724 Paris Cedex 15, France and julia.chamot-rooke@pasteur.fr.
authors.forename: Julia
authors.initials: J
authors.lastname: Chamot-Rooke
authors.affiliation: From the Molecular Genetics Unit, CNRS ERL 3526, ghh2001@med.cornell.edu.
authors.forename: Gerard H M
authors.initials: GH
authors.lastname: Huysmans
mesh_terms: D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017386:Sequence Homology, Amino Acid
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D050505:Mutant Proteins; C560058:PulD protein, E coli
keywords: bacteria; hydrogen exchange mass spectrometry; membrane protein; protein folding; secretion