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, not least to inform efforts for the therapeutic modulation of LLPS phenomena. Given the rapidly increasing number of biologically and disease-relevant condensate systems, experimental techniques that enable high-throughput analysis of protein phase behaviour are required. Here, we present a droplet microfluidic platform, termed PhaseScan, for the rapid generation of protein phase diagrams, a fundamental measure with which to characterise protein phase behaviour in chemical space. 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 throughout and reduced sample consumption with respect to conventional experiments. We find that the phase boundary at which FUS transitions from a one-phase to a two-phase state is modulated by the small molecule 1,6-hexanediol, and estimate the free-energy landscape of this system using Flory–Huggins theory. Our study thus provides a basis for the rapid acquisition of phase diagrams through the application of microdroplet techniques and paves the way for a wide range of applications, enabling swift characterisation of the effect of environmental conditions and coacervate species on the thermodynamics of phase separation.
Competing Interest Statement
Parts of this work have been the subject of a patent application filed by Cambridge Enterprise Limited, a fully owned subsidiary of the University of Cambridge.