TY - JOUR

T1 - Capillary flow experiments for thermodynamic and kinetic characterization of protein liquid-liquid phase separation

AU - Stender, Emil G.P.

AU - Ray, Soumik

AU - Norrild, Rasmus K.

AU - Larsen, Jacob Aunstrup

AU - Petersen, Daniel

AU - Farzadfard, Azad

AU - Galvagnion, Céline

AU - Jensen, Henrik

AU - Buell, Alexander K.

N1 - Funding Information: Charlotte O’Shea from University of Copenhagen is thanked for donating the TEV-MBP plasmid. We thank Dr. Tim Nott (Department of Biochemistry) and Dr. Andrew Baldwin (Department of Chemistry), both at Oxford University, for donating the PET-30M plasmids containing the GST-Ddx4n1 fusion constructs and for help with protein production. The DTU bio-imaging core (at DTU bioengineering) is acknowledged for confocal imaging and FRAP experiments. Kristina Mielec is thanked for technical assistance. E.G.P.S, R.K.N, J.A.L., and A.K.B. would like to acknowledge the Novo Nordisk Foundation for funding (Grant number: NNFSA170028392). S.R. and A.K.B. would like to acknowledge VILLUM FONDEN for financial support (Grant number 35823). C.G. thanks the Lundbeck Foundation (Grant number: R314-2018-3493) and C.G. and D.P. thank the Carlsberg Foundation (Grant number: CF19-0382). The Carlsberg Foundation is also acknowledged for funding the FIDA 1 instrument with a grant to Andreas H. Lausten (Grant number: CF19-0055). Funding from Novo Nordisk Foundation (grant NNFOC0055625) for the infrastructure “Imaging microbial language in biocontrol (IMLiB)” is acknowledged. We would like to thank Lars Boyens-Thiele for assistance with experiments. Publisher Copyright: © 2021, The Author(s).

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

U2 - 10.1038/s41467-021-27433-y

DO - 10.1038/s41467-021-27433-y

M3 - Journal article

C2 - 34911929

AN - SCOPUS:85121391953

VL - 12

JO - Nature Communications

JF - Nature Communications

SN - 2041-1723

IS - 1

M1 - 7289

ER -