Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

Emil G.P. Stender, Soumik Ray, Rasmus K. Norrild, Jacob Aunstrup Larsen, Daniel Petersen, Azad Farzadfard, Céline Galvagnion, Henrik Jensen, Alexander K. Buell*

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Liquid-liquid phase separation or LLPS of proteins is a field of mounting importance and the value of quantitative kinetic and thermodynamic characterization of LLPS is increasingly recognized. We present a method, Capflex, which allows rapid and accurate quantification of key parameters for LLPS: Dilute phase concentration, relative droplet size distributions, and the kinetics of droplet formation and maturation into amyloid fibrils. The binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds can also be determined. We apply Capflex to characterize the LLPS of Human DEAD-box helicase-4 and the coacervate system ssDNA/RP3. Furthermore, we study LLPS and the aberrant liquid-to-solid phase transition of α-synuclein. We quantitatively measure the decrease in dilute phase concentration as the LLPS of α-synuclein is followed by the formation of Thioflavin-T positive amyloid aggregates. The high information content, throughput and the versatility of Capflex makes it a valuable tool for characterizing biomolecular LLPS.
Original languageEnglish
Article number7289
JournalNature Communications
Volume12
Number of pages18
ISSN2041-1723
DOIs
Publication statusPublished - 2021

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