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

Abstract

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 19F NMR reports on the formation and evolution of both phases throughout the sample, including their concentrations and volumes. Meanwhile, spatially-selective 19F 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.

Formation of versus Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity

“…This use of a fluorine-containing additive is distinct from the method we describe here. The main goal of the studies in ref was to characterize the kinetics of phase separation. The authors used 19 F chemical shift data to measure BSA concentration in the condensed phase; these measurements required an external calibration curve constructed with samples containing independently determined BSA concentrations.…”

“…While our manuscript was under review, a paper appeared describing the use of 2,2,2-trifluoroethanol (TFE) as a probe to study phase separation induced by addition of YCl 3 to concentrated solutions of bovine serum albumin (BSA) via 19 F NMR. 56 This use of a fluorine-containing additive is distinct from the method we describe here. The main goal of the studies in ref 56 was to characterize the kinetics of phase separation.…”

Liquid–Liquid Phase Separation of an Intrinsically Disordered Region of a Germ Cell-Specific Protein Modulates the Stability and Conformational Exchange Rate of SH3 Domain

“…Getting information about the enveloped biomolecules in phase separated droplets at the atomic level has always been a challenge. Bramham et al used a small probe to characterize the kinetics of protein LLPS by using 19 F NMR . In this paper, we have shown that 19 F-NMR is a powerful tool for studying biomacromolecules inside the droplets.…”

“…Bramham et al used a small probe to characterize the kinetics of protein LLPS by using 19 F NMR. 39 In this paper, we have shown that 19 F-NMR is a powerful tool for studying biomacromolecules inside the droplets. Moreover, our finding has general implications in the field of LLPS.…”

Multinuclear 1D & 2D NMR with 19F-Photo-CIDNP hyperpolarization in a microfluidic chip with broadband microcoil

“…Uniquely, we now exploit two high-band channels in the single microcoil set-up, permitting the synchronous application of 1 H and 19 F Larmor frequencies within one pulse sequence. 19 F is a 100% naturally abundant NMR active (I=1/2) nuclide which finds many applications in supramolecular, 49 (bio)materials, 50 and medicinal chemistry, 51 among others. Moreover, the 19 F nuclides holds promise for photo-CIDNP experiments because of the often large electron-nucleus hyperfine coupling that determines hyperpolarization efficiencies.…”