Coalescence-Driven Local Crowding Promotes Liquid-to-Solid-Like Phase Transition in a Homogeneous and Heterogeneous Droplet Assembly

Liquid-to-solid-like phase transition (LSPT) of disordered proteins via metastable liquid-like droplets is a well-documented phenomenon in biology and linked to many pathological conditions including neurodegenerative diseases. However, very less is known about the early microscopic events and transient intermediates involved in the irreversible protein aggregation of functional globular proteins. Herein, using a range of microscopic and spectroscopic techniques, we show that the LSPT of a functional globular protein, human serum albumin (HSA) is exclusively driven by spontaneous coalescence of liquid-like droplets involving various transient intermediates in a temporal manner. We show that inter-droplet communication via coalescence is essential for both nucleation and growth of amorphous aggregates within individual droplets, which subsequently transform to amyloid-like fibrils. Immobilized droplets neither show any nucleation nor any growth upon aging. Moreover, we found that exchange of materials with the dilute dispersed phase has negligible influence on the LSPT of HSA. Notably, binding of small ligands modulates the feasibility and kinetics of LSPT of HSA, suggesting a possible regulatory mechanism that cells utilize to control the dynamics of LSPT. Further, using a dynamic heterogeneous droplet assembly of two functional proteins, HSA and transferrin (Tf), we show an intriguing phenomenon within the fused droplets where both liquid-like and solid-like phases co-exist within the same droplet, which eventually transform to a mixed fibrillar assembly. These microscopic insights not only highlight the importance of inter-droplet interactions behind the LSPT of biomolecules but also showcase its adverse effect on the structure and function of other functional proteins in a crowded and heterogeneous protein assembly.