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Proc Natl Acad Sci U S A. 2017 Mar 7;114(10):2598-2603. doi: 10.1073/pnas.1616576114. Epub 2017 Feb 21.

Membrane integration of an essential β-barrel protein prerequires burial of an extracellular loop.

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Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138.
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138;
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115.


The Bam complex assembles β-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. These proteins comprise cylindrical β-sheets with long extracellular loops and create pores to allow passage of nutrients and waste products across the membrane. Despite their functional importance, several questions remain about how these proteins are assembled into the OM after their synthesis in the cytoplasm and secretion across the inner membrane. To understand this process better, we studied the assembly of an essential β-barrel substrate for the Bam complex, BamA. By mutating conserved residues in the β-barrel domain of this protein, we generated three assembly-defective BamA substrates that stall early in the folding process in the periplasm. Two of the three defective substrates, which harbor mutations within β-strands, fail to associate productively with the Bam complex. The third substrate, which harbors mutations in a conserved extracellular loop, accumulates on BamD during assembly, but does not integrate efficiently into the membrane. The assembly of all three substrates can be restored by artificially tethering a region of the substrate, which ultimately becomes an extracellular loop, to the lumen of the forming β-barrel. These results imply that a critical step in the folding process involves the interaction of residues on the interior of the nascent β-barrel wall with residues in one of the extracellular loops. We conclude that a prerequisite for membrane integration of β-barrel proteins is burial of the extracellular loops within the forming β-barrel.


Bam complex; outer membrane; protein folding; β-barrel

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