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Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.

ABSTRACT: Unfolded outer membrane beta-barrel proteins have been shown to self-associate in the absence of lipid bilayers. We previously investigated the formation of high molecular weight species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native ?-sheet structure. We have further probed the conformation of self-associated OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross-? a structure inherent in amyloid fibrils, and by observing the species by electron microscopy. The significant increase in fluorescence indicative of Thioflavin T binding and the appearance of fibrillar species by electron microscopy verify that the protein forms amyloid-like fibril structures upon oligomerization. These results are also consistent with our previous kinetic analysis of OmpA self-association that revealed a nucleated growth polymerization mechanism, which is frequently observed in amyloid formation. The discovery of OmpA's ability to form amyloid-like fibrils provides a new model protein with which to study fibrillization, and implicates periplasmic chaperone proteins as capable of inhibiting fibril formation.

SUBMITTER: Danoff EJ

PROVIDER: S-EPMC4509890 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.

Danoff Emily J EJ Fleming Karen G KG

PloS one 20150721 7

Unfolded outer membrane beta-barrel proteins have been shown to self-associate in the absence of lipid bilayers. We previously investigated the formation of high molecular weight species by OmpA, with both the transmembrane domain alone and the full-length protein, and discovered that the oligomeric form contains non-native β-sheet structure. We have further probed the conformation of self-associated OmpA by monitoring binding to Thioflavin T, a dye that is known to bind the cross-β a structure ...[more]

PMID: 26196893

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Project description:OmpA is one of only a few transmembrane proteins whose folding and stability have been investigated in detail. However, only half of the OmpA mass encodes its transmembrane β-barrel; the remaining sequence is a soluble domain that is localized to the periplasmic side of the outer membrane. To understand how the OmpA periplasmic domain contributes to the stability and folding of the full-length OmpA protein, we cloned, expressed, purified and studied the OmpA periplasmic domain independently of the OmpA transmembrane β-barrel region. Our experiments showed that the OmpA periplasmic domain exists as an independent folding unit with a free energy of folding equal to -6.2 (±0.1) kcal mol(-1) at 25°C. Using circular dichroism, we determined that the OmpA periplasmic domain adopts a mixed alpha/beta secondary structure, a conformation that has previously been used to describe the partially folded non-native state of the full-length OmpA. We further discovered that the OmpA periplasmic domain reduces the self-association propensity of the unfolded barrel domain, but only when covalently attached (in cis). In vitro folding experiments showed that self-association competes with β-barrel folding when allowed to occur before the addition of membranes, and the periplasmic domain enhances the folding efficiency of the full-length protein by reducing its self-association. These results identify a novel chaperone function for the periplasmic domain of OmpA that may be relevant for folding in vivo. We have also extensively investigated the properties of the self-association reaction of unfolded OmpA and found that the transmembrane region must form a critical nucleus comprised of three molecules before undergoing further oligomerization to form large molecular weight species. Finally, we studied the conformation of the unfolded OmpA monomer and found that the folding-competent form of the transmembrane region adopts an expanded conformation, which is in contrast to previous studies that have suggested a collapsed unfolded state.

Dataset Information

Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.

Publications

Aqueous, Unfolded OmpA Forms Amyloid-Like Fibrils upon Self-Association.

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