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

Abstract 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 of 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.