Tuning Functional Amyloid Formation Through Disulfide Engineering.

Frontiers in Microbiology
Anthony BalistreriMatthew R Chapman

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. Amyloi...Continue Reading

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Citations

Jan 2, 2021·Colloids and Surfaces. B, Biointerfaces·Esra YucaUrartu Özgür Şafak Şeker
Dec 2, 2021·ACS Synthetic Biology·Sol Vendrell-FernándezRafael Giraldo

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Methods Mentioned

BETA
protein folding
gel filtration
Electrophoresis
Assay
size exclusion chromatography
circular dichroism
transmission electron microscopy

Software Mentioned

CsgA CC
ImageJ

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