Investigating strategies for creating cross-linked amyloid fibril networks through branching of amyloid growth

Hydrogel biomaterials have been extensively explored for applications in medicine, materials science, and the development of functionalized materials. Traditionally, hydrogels were produced using simple polymers, but advancements over recent decades have enabled the use of biological materials such as proteins, peptides, polysaccharides, and even amyloid fibrils. Among these, amyloid-based hydrogels have demonstrated unique advantages, including enhanced cell adhesion and differentiation. Furthermore, they can be engineered as living materials using bacteria capable of producing and repairing the hydrogel in situ. Here we investigate novel strategies for controlling amyloid fibrillation using the functional amyloid CsgA. We designed fusion proteins combining two CsgA moieties to explore methods for creating branched fibril networks. Our approach utilized two distinct strategies: passive and active branching. The passive strategy involved direct fusion of two CsgA moieties separated by a designed alpha-helical linker and engineered to integrate into fibrils without external intervention. The active branching approach incorporated a redox-sensitive CsgA variant containing an internal disulfide bridge that blocks fibrillation until reduced. This design allows for precise control of amyloid fibrillation in the active variants. We analyzed these constructs qualitatively approach using a combination of transmission electron microscopy (TEM), real-time atomic force microscopy (AFM), and total internal reflection fluorescence (TIRF) microscopy, supported by quantitative image analysis. While we did not observe direct evidence of fibril branching, our modifications led to significant changes in fibrillation behavior. Notably, TIRF imaging revealed a marked increase in high-density fibril regions following the activation of our engineered constructs, indicating the potential for controlled assembly of higher-order structures. These findings provide new insights into controlling amyloid fibrillation and suggest alternative strategies for manipulating fibril organization. The observed ability to alter local fibril density through chemical triggers offers promising directions for developing responsive biomaterials. We propose refinements for future design and suggest new directions to optimize amyloid-based hydrogels for next-generation biomaterial applications.