Functional amyloids are integral to bacterial biofilms, yet the molecular details of their folding remain poorly understood. We performed extensive all-atom molecular dynamics (MD) and steered MD (SMD) simulations to dissect the folding mechanism of the Pseudomonas amyloid protein FapC, the primary scaffold of biofilm integrity. Starting from AlphaFold2-derived monomers, three independent 1-μs simulations captured stable β-strand cores while allowing disordered loops to relax and sample conformational space. Complementary simulations of NMR-derived disordered ensembles (9 μs total) revealed repeat-specific β-sheet nucleation, with R2 and R3 acting as dominant folding hotspots that seeded formation of a nascent monomeric fold. To probe the reverse process, we carried out 8.4 μs of SMD simulations that systematically unfolded FapC. These trajectories uncovered a five-step sequence of β-sheet and interlayer separations. Strikingly, the order of unfolding mirrored the sequence of folding events, with the same repeat regions acting as key determinants of both forward and reverse transitions. Together, these results establish an atomistic framework for FapC folding, in which repeat-driven β-sheet nucleation propagates toward structured monomeric intermediates. By integrating unbiased and steered MD, we delineate the reciprocal pathways of folding and unfolding, providing mechanistic insight into the assembly of a functional amyloid protein.
Gölcük et al. (Sun,) studied this question.