Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC

ABSTRACT Bacterial functional amyloids are evolutionarily optimized to aggregate to help them fulfil their biological functions, e.g. to provide mechanical stability to biofilm. Amyloid is formed in Pseudomonas sp. by the protein FapC which contains 3 imperfect repeats connected by long linkers. Stepwise removal of these repeats slows down aggregation and increases the propensity of amyloids to fragment during the fibrillation process, but how these mechanistic properties link to fibril stability is unclear. Here we address this question. The extreme robustness of functional amyloid makes them resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. To quantify this, we first measured the denaturing potency of FA using 3 small acid-resistant proteins (S6, lysozyme and ubiquitin). This revealed a linear relationship between [FA] and the free energy of unfolding with a slope of m FA , as well as a robust correlation between protein residue size and m FA . We then measured the solubilisation of fibrils formed from different FapC variants (with varying number of repeats) as a function of [FA]. The resulting m FA values revealed a decline in the number of residues driving amyloid formation when at least 2 repeats were deleted. The midpoint of denaturation declined monotonically with progressive removal of repeats and correlated with solubility in SDS. Complete removal of all repeats led to fibrils which were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one imperfect repeat is required for the stability of functional amyloid.