Temporal and spatial characterisation of protein liquid-liquid phase separation using NMR spectroscopy.

Liquid-liquid phase separation (LLPS) of protein solutions is increasingly recognised as an important phenomenon in cell biology and biotechnology. However, opalescence and concentration fluctuations render LLPS difficult to study, particularly when characterising the kinetics of the phase transition and layer separation. Here, we demonstrate the use of a probe molecule trifluoroethanol (TFE) to characterise the kinetics of protein LLPS by NMR spectroscopy. The chemical shift and linewidth of the probe molecule are sensitive to local protein concentration, with this sensitivity resulting in different characteristic signals arising from the dense and lean phases. Monitoring of these probe signals by conventional bulk-detection 19 F NMR reports on the formation and evolution of both phases throughout the sample, including their concentrations and volumes. Meanwhile, spatially-selective 19 F NMR, in which spectra are recorded from smaller slices of the sample, was used to track the distribution of the different phases during layer separation. This experimental strategy enables comprehensive characterisation of the process and kinetics of LLPS, and may be useful to study phase separation in protein systems as a function of their environment.