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 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 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.
Highlights
In transmembrane proteins, the amino acid residues that reside in the membrane interior generally possess hydrophobic side chains in order to have an energetically favorable interaction with the nonpolar lipid acyl chains [1]
We have shown that the UAQ transmembrane domain of Outer Membrane Protein A (OmpA) self-associates in low denaturant conditions lacking lipid bilayers to form high molecular weight species that exhibit substantial β-sheet structure, are capable of binding Thioflavin T, and appear as long flexible fibrils by transmission electron microscopy (TEM), leading to the conclusion that UAQ OmpA forms amyloid-like fibrils
It is likely that other outer membrane proteins (OMPs) form such structures, as high molecular weight species have been observed for a number of OMPs in the absence of membranes [6]
Summary
The amino acid residues that reside in the membrane interior generally possess hydrophobic side chains in order to have an energetically favorable interaction with the nonpolar lipid acyl chains [1] This results in a high propensity for aggregation in the absence of the bilayer environment, especially for α-helical membrane proteins, which contain continuous stretches of hydrophobic residues; this explains the common need for detergents when purifying these proteins. Β-barrel outer membrane proteins (OMPs) offer the advantage of a lower aggregation propensity due to their sequence pattern of alternating hydrophobic and hydrophilic residues—a consequence of only every other residue in the transmembrane β-strands facing the lipid environment [2] It can be seen that in each strand the side chains are oriented in alternating directions and PLOS ONE | DOI:10.1371/journal.pone.0132301 July 21, 2015
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