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 and Alexander K. Buell ()
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Emil G. P. Stender: Technical University of Denmark
Soumik Ray: Technical University of Denmark
Rasmus K. Norrild: Technical University of Denmark
Jacob Aunstrup Larsen: Technical University of Denmark
Daniel Petersen: Faculty of Health and Medical Sciences, University of Copenhagen
Azad Farzadfard: Technical University of Denmark
Céline Galvagnion: Faculty of Health and Medical Sciences, University of Copenhagen
Henrik Jensen: FIDA Biosystems Aps
Alexander K. Buell: Technical University of Denmark

Nature Communications, 2021, vol. 12, issue 1, 1-18

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-27433-y

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