Early Pathway Selection Precedes Conformational Locking in Protein Self-Assembly

Abstract

When early intermediates begin to bias subsequent pathways during protein self-assembly remains difficult to resolve experimentally because early-stage processes are transient and poorly accessible. Here, using regenerated silk fibroin as a model system, we experimentally capture and analyze an early nucleation window in which assembly pathways begin to diverge before conformational locking occurs. By combining controlled electric-field perturbation with multiscale structural characterization and coarse-grained simulations, we identify reproducible nanoscale intermediates that mediate pathway selection. Within this window, electric fields bias early assembly kinetics in a nonmonotonic manner, modulating the formation, elongation, and competition of intermediates rather than directly enforcing structure or orientation. Time-resolved measurements show that subsequent growth proceeds primarily through elongation and fusion of existing intermediates, while β-sheet formation and crystallization emerge only at later stages and serve to stabilize pathways that were biased at earlier stages. Together, we locate the emergence of detectable pathway biasing within the nucleation stage and provide a framework for understanding and guiding protein self-assembly under nonequilibrium conditions.