Phase separation kinetics of bio-inspired condensates under passive and active conditions
Résumé
We investigate the structural dynamics and kinetics of condensates made up of cationic peptides (RRASLRRASL) and poly uracil (polyU) RNAs, serving as an ideal model system for membrane-less organelles. Employing absorbance measurements at 500 nm, the critical peptide concentration is estimated to be 400 µM at a fixed polyU concentration (0.5 g/L) in a physiological buffer (50 mM HEPES pH 7.4). Confocal microscopy images of fluorescently labelled peptide/polyU condensate at this concentration display a droplet-like morphology (1 to 5 µm). The absorbance increased rapidly (t < 1s) and remained stable for 150 minutes for the sample containing 0.5 g/L polyU and 1000 µM peptide, indicating coacervation. The charge ratio was estimated to be 2.45. Preliminary experiments were carried out by performing time-resolved small-angle X-ray scattering (TR-SAXS) with a stopped-flow device for rapid mixing at 37 •C. TR-SAXS patterns revealed that with unphosphorylated peptides, the condensate size increased rapidly and reached a few hundred nanometers in t < 400 ms. Contrary to the anticipated α ≈ for a standard coalescence process established from the scaling law R∼tˆα, our empirical findings yielded a scaling exponent of 0.45 ± 0.03, marking a 35% deviation from the theoretical prediction. Next, we studied the dephosphorylation of double-phosphorylated peptides by Lambda Protein Phosphatase (LPP), which dephosphorylates the serine residues of the peptide, thus driving coacervation. The absorbance linearly increased by 50% within 150 minutes for the sample containing 0.5 g/L polyU, 1000 µM peptide, and 400 U/mL LPP, denoting an increase in the number and/or size of the condensates over time. When the sample concentration was doubled, the absorbance started to increase at t = 15 minutes and remained stable for the subsequent 125 minutes. This could be attributed to the higher sample concentration, which increased the condensation rate, resulting in an early equilibrium.
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