Abstract
Bacterial functional amyloids are evolutionarily optimized to aggregate, so much so that the extreme robustness of functional amyloid makes it very difficult to examine their structure-function relationships in a detailed manner. Previous work has shown that functional amyloids are resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. However, systematic investigation requires a quantitative analysis of FA's ability to denature proteins. Amyloid formed by Pseudomonas sp. protein FapC provides an excellent model to investigate FA denaturation. It contains three imperfect repeats, and stepwise removal of these repeats slows fibrillation and increases fragmentation during aggregation. However, the link to stability is unclear. We first calibrated FA denaturation using three small, globular, and acid-resistant proteins. This revealed a linear relationship between the concentration of FA and the free energy of unfolding with a slope of mFA+pH (the combined contribution of FA and FA-induced lowering of pH), as well as a robust correlation between protein size and mFA+pH We then measured the solubilization of fibrils formed from different FapC variants with varying numbers of repeats as a function of the concentration of FA. This revealed a decline in the number of residues driving amyloid formation upon deleting at least two repeats. The midpoint of denaturation declined with the removal of repeats. Complete removal of all repeats led to fibrils that were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one repeat is required for the stability of functional amyloid.
Highlights
The term ‘amyloid’ is normally associated with misfolding of different proteins, resulting in neurodegenerative diseases like Alzheimer’s and Parkinson’s disease, but the number of cases where the amyloid structure is used for functional purposes is steadily increasing[1, 2]
We considered the alternative option of assuming that the equilibrium constant corresponds to the concentration of free protein, inspired by the use of free monomer concentration to determine the stability of Aβ fibrils[25] as well as other fibril stability studies[26]; this approach does not yield a linear relationship between the free energy and [formic acid (FA)] and does not allow us to extrapolate robustly to 0 M FA
We have quantitated the denaturing potency of formic acid against globular proteins using thermal scans and isothermal titration monitored by near-UV circular dichroism (CD)
Summary
The term ‘amyloid’ is normally associated with misfolding of different proteins, resulting in neurodegenerative diseases like Alzheimer’s and Parkinson’s disease, but the number of cases where the amyloid structure is used for functional purposes is steadily increasing[1, 2]. The first functional amyloid to be identified and purified were the curli fibrils expressed by E. coli and Salmonella enteritidis[3, 4]. Since their discovery, curli has been shown to be widely expressed among different bacteria, spanning at least four different phyla[5]. Four additional Csg proteins are expressed from the csgDEFG operon and they act as a transcription regulator of the csgBAC operon (CsgD), chaperones (CsgE/CsgF) and as an outer membrane pore protein (CsgG)[9,10,11]. All Csg proteins, except CsgD, are targeted for Sec-dependent secretion across the inner bacterial membrane to the periplasm[12]
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