RT Journal Article
SR Electronic
T1 Rapid Generation of Protein Condensate Phase Diagrams Using Combinatorial Droplet Microfluidics
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.06.04.132308
DO 10.1101/2020.06.04.132308
A1 William E. Arter
A1 Runzhang Qi
A1 Georg Krainer
A1 Timothy J. Welsh
A1 Yufan Xu
A1 Peter St George-Hyslop
A1 Simon Alberti
A1 Tuomas P.J. Knowles
YR 2020
UL http://biorxiv.org/content/early/2020/06/10/2020.06.04.132308.abstract
AB 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.Competing Interest StatementParts 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.