1. Prepore Stability Controls Productive Folding of the BAM-independent Multimeric Outer Membrane Secretin PulD.
- Author
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Guilvout I, Brier S, Chami M, Hourdel V, Francetic O, Pugsley AP, Chamot-Rooke J, and Huysmans GH
- Subjects
- Amino Acid Sequence, Bacterial Outer Membrane Proteins genetics, Escherichia coli growth & development, Escherichia coli Proteins genetics, Mutagenesis, Site-Directed, Mutant Proteins genetics, Mutation genetics, Protein Binding, Protein Conformation, Protein Multimerization, Protein Stability, Sequence Homology, Amino Acid, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins metabolism, Escherichia coli metabolism, Escherichia coli Proteins chemistry, Escherichia coli Proteins metabolism, Mutant Proteins chemistry, Mutant Proteins metabolism, Protein Folding
- 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 N
3 domain 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 N3 domain stability, suggested that the N3 domain 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., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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