Phase transitions are ubiquitous in nature, taking part in the formation of both synthetic and living materials. In cells, biomolecule solutions can undergo liquid-liquid phase separation but also various liquid to solid-like transitions (i.e. aggregation and gelation) that regulate biochemical processes. Uncovering the assembly phase behavior of one-component protein solutions in vitro is the first step toward understanding the non-equilibrium behavior of multicomponent solutions in living organisms. Globular and intrinsically disordered protein solutions are relatively well understood and conceptualized via colloidal and polymer theories that highlight the role of electrostatic interactions. However, solutions of multidomain proteins containing both folded and intrinsically disordered regions (IDRs) are less studied.
To fill this gap, we investigate the assembly phase behavior of an in vitro solution of an engineered recombinant silk-like protein, NT-2Rep-CT, containing two folded terminal domains connected by an IDR tether. We showed by light microscopy and analytical ultracentrifugation measurements that the protein solution can exist in multiple material states including homogenous dilute solutions, metastable liquid-liquid coexistence, and irreversible aggregates. The experiments highlight the challenge in identifying equilibrium states but also demonstrate the critical role of metastability of the solution and its aging on the properties of the final material. To understand and generalize the findings, we have considered the thermodynamics of the system and proposed a minimalist phase diagram for the assembly phase response of the NT-2Rep-CT protein. The work paves way toward a generalized understanding of in vitro protein solutions.
[1] Fedorov, Escalona, Tolmachev, Harmat, Scacchi, Sammalkorpi, Aranko, Linder, Small 20 2306817 (2024).
[2] Fedorov*, Sammalisto*, Harmat*, Ahlberg, Koskela, Haataja, Scacchi, Sammalkorpi, Linder, Advanced Functional Materials 35 2410421 (2025).