Tuning Functional Amyloid Formation Through Disulfide Engineering.

Area of Science:

• Biochemistry
• Microbiology
• Structural Biology

Background:

• Functional amyloid fibers, like curli produced by Enterobacteriaceae, are vital protein polymers in cellular biology and biofilm formation.
• CsgA is the primary subunit of curli, rapidly forming amyloid structures in vitro, which presents challenges for studying its aggregation process.

Purpose of the Study:

• To engineer a CsgA variant enabling tunable amyloid formation controlled by the protein's redox state.
• To investigate the potential of disulfide bond manipulation for controlling amyloidogenic protein aggregation.

Main Methods:

• Engineered a double cysteine variant of CsgA (CsgA_CC) to modulate amyloid formation.
• Characterized CsgA_CC's amyloid formation under varying oxidizing and reducing conditions.
• Utilized thioflavin T fluorescence assays to compare CsgA_CC amyloid kinetics with wild-type CsgA (CsgA WT).

Main Results:

• Oxidized CsgA_CC remained soluble and non-amyloid for over 90 days, unlike CsgA WT.
• Addition of reducing agents induced CsgA_CC amyloid formation within hours, yielding fibers indistinguishable from CsgA WT.
• Amyloid formation kinetics of reduced CsgA_CC mirrored CsgA WT, and could be reversibly halted by oxidation.

Conclusions:

• CsgA_CC provides a novel tool for controlling amyloid formation via redox state manipulation.
• This engineered variant demonstrates the feasibility of using convertible disulfide bonds to regulate amyloidogenic protein aggregation.
• The findings offer a proof-of-concept for developing new strategies to study and control functional amyloid formation.