Single-molecule mass measurements reveal distinct effects of sodium and potassium on mini-spidroin assembly

Spider silk formation involves tightly regulated protein assembly influenced by pH and the presence of ions. Kosmotropic salts induce phase separation of spidroins, however, their exact role in assembly is not clear. Here, we investigate how sodium and potassium phosphate affect spidroin interactions via the single-molecule method mass photometry. We observed that spidroin oligomerization occurs at low nanomolar protein concentrations. Potassium ions were found to stabilize a compact conformation of individual spidroins and slow down pH-induced β-sheet aggregation, consistent with its more kosmotropic nature. Microfluidic MP showed that pre-assembly of the protein through salt-induced phase separation reduced the number and size of oligomeric intermediates that form upon acidification. Together, the findings suggest that spidroins have an inherent ability to self-assemble, blurring the line between one- and two-phase status. Subtle differences in ion composition are sufficient to change spidroin stability and assembly, potentially contributing to silk spinning in vivo by balancing storage stability with rapid fiber formation.