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

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Listed:
  • 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)

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.

Suggested Citation

  • Emil G. P. Stender & Soumik Ray & Rasmus K. Norrild & Jacob Aunstrup Larsen & Daniel Petersen & Azad Farzadfard & Céline Galvagnion & Henrik Jensen & Alexander K. Buell, 2021. "Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation," Nature Communications, Nature, vol. 12(1), pages 1-18, December.
    • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-27433-y
    DOI: 10.1038/s41467-021-27433-y
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