# pH-triggered clustering regulates β-sheet activation in silk assembly

## TL;DR

The study reveals how pH changes control the step-by-step formation of silk's strong structure through clustering and sheet assembly.

## Contribution

A new method combining neutron scattering and fluorescence tracks silk's hierarchical assembly in real time.

## Key findings

- pH-driven gelation involves nanoscale clustering and domain growth before β-sheet formation.
- Methanol-induced gelation skips intermediates and causes rapid aggregation.
- NUrF provides a strategy to study hierarchical assembly in protein materials.

## Abstract

Silk fibres derive their exceptional properties from hierarchical protein organisation, yet the molecular pathways that guide this structural transformation remain poorly resolved. During regenerated silk fibroin gelation under biomimetic gradual acidification, we identify a stepwise assembly pathway comprising nanoscale clustering, domain growth within clusters, and mesoscale network formation. Time-resolved small-angle neutron scattering performed simultaneously with turbidity and fluorescence emission (NUrF) identifies unique intermediates and a regulated onset of β-contacts and β-sheets assembly, indicating that fibril formation requires prior compaction and network connectivity. By contrast, methanol-induced gelation bypasses these intermediates, driving rapid aggregation. These findings define the sequence and timing of events that construct silk’s hierarchical architecture without accidental aggregation, showing how pathway selection governs material outcomes. This multiscale resolution achieved by NUrF provides a broadly applicable strategy for probing hierarchical assembly in silk and other protein materials.

Silk fibres derive their exceptional properties from hierarchical protein organisation, but the underlying molecular pathways remain underexplored. Here, the authors combine time-resolved small-angle neutron scattering, pH and turbidity measurements, and fluorescence emission to identify a stepwise assembly pathway comprising nanoscale clustering, domain growth within clusters, and mesoscale network formation.