Abstract
Many organisms produce “functional” amyloid fibers, which are stable protein polymers that serve many roles in cellular biology. Certain Enterobacteriaceae assemble functional amyloid fibers called curli that are the main protein component of the biofilm extracellular matrix. CsgA is the major protein subunit of curli and will rapidly adopt the polymeric amyloid conformation in vitro. The rapid and irreversible nature of CsgA amyloid formation makes it challenging to study in vitro. Here, we engineered CsgA so that amyloid formation could be tuned to the redox state of the protein. A double cysteine variant of CsgA called CsgACC was created and characterized for its ability to form amyloid. When kept under oxidizing conditions, CsgACC did not adopt a β-sheet rich structure or form detectable amyloid-like aggregates. Oxidized CsgACC remained in a soluble, non-amyloid state for at least 90 days. The addition of reducing agents to CsgACC resulted in amyloid formation within hours. The amyloid fibers formed by CsgACC were indistinguishable from the fibers made by CsgA WT. When measured by thioflavin T fluorescence the amyloid formation by CsgACC in the reduced form displayed the same lag, fast, and plateau phases as CsgA WT. Amyloid formation by CsgACC could be halted by the addition of oxidizing agents. Therefore, CsgACC serves as a proof-of-concept for capitalizing on the convertible nature of disulfide bonds to control the aggregation of amyloidogenic proteins.
Highlights
Amyloids are fibril aggregates of proteins which have misfolded to form a characteristic cross β-sheet secondary structure (Chiti and Dobson, 2006)
To control amyloid aggregation by CsgA we hypothesized that strategically-placed cysteine residues would allow the generation of a CsgA molecule that could be locked in a non-amyloid state
Both CsgA WT and CsgACC that was incubated with the reducing agent TCEP displayed a marked decrease in SDS solubility over a 48 h period (Figure 2A)
Summary
Amyloids are fibril aggregates of proteins which have misfolded to form a characteristic cross β-sheet secondary structure (Chiti and Dobson, 2006). The fibers deposit in cells in dense bodies known as plaques and are implicated in many well-known neurodegenerative diseases including Alzheimer, Parkinson, and Huntington Disease (Chiti and Dobson, 2017) For this reason, the nature of amyloids and how they form is an active area of research. Though the disease causing amyloids are composed of misfolded proteins, there are many examples of “functional amyloids” that organisms create for a predetermined purpose (Chapman et al, 2002; Otzen, 2010). These fibers are composed of proteins that adopt the amyloid fold not by misfolding, rather they do so intentionally and efficiently (Deshmukh et al, 2018). Functional amyloids provide researchers with well-defined and adaptable models for studying the basic tenets of amyloid formation
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