Rapid Generation of Protein Condensate Phase Diagrams Using Combinatorial Droplet Microfluidics

Abstract

The assembly of intracellular proteins into biomolecular condensates via liquid–liquid phase separation (LLPS) has emerged as a fundamental process underlying the organisation and regulation of cellular space and function. Physicochemical characterisation of the parameters that control and modulate phase separation is therefore essential for an improved understanding of protein phase behaviour, including for the therapeutic modulation of LLPS phenomena. A fundamental measure with which to describe protein phase behaviour in chemical space is the phase diagram. Characterisation of phase diagrams requires measuring the presence or absence of the condensed phase under a multitude of conditions and, as such, is associated with significant consumption of time and sample volume even when performed in microwell format. However, due to the rapidly increasing number of biologically and disease-relevant condensate systems, experimental techniques that enable high-throughput analysis of protein phase behaviour are required. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for the rapid and high-resolution acquisition of protein phase diagrams. Using this platform, we demonstrate characterisation of the phase behaviour of a pathologically relevant mutant of the protein fused in sarcoma (FUS) in a highly parallelised manner, with significantly improved assay throughput and reduced sample consumption. We demonstrate the capability of the platform by finding the phase boundary at which FUS transitions from a one-phase to a two-phase state as modulated by 1,6-hexanediol, and estimate the free-energy landscape of this system using Flory–Huggins theory. These results thus provide a basis for the rapid acquisition of phase diagrams through the application of microdroplet techniques and pave the way for a wide range of applications, enabling rapid characterisation of the effect of environmental conditions and coacervate species on the thermodynamics of phase separation.