Biomolecular phase separation through the lens of sodium‐23 NMR

Abstract

Phase separation is a fundamental physicochemical process underlying the spatial arrangement and coordination of cellular events. Detailed characterization of biomolecular phase separation requires experimental access to the internal environment of dilute and especially condensed phases at high resolution. In this study, we take advantage from the ubiquitous presence of sodium ions in biomolecular samples and present the potentials of 23Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of 23Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that 23Na NMR probes of rotational and translational mobility of sodium ions are capable of capturing the increasing levels of confinement in agarose gels in dependence of agarose concentration. The 23Na NMR approach is then applied to a gel‐forming phenylalanine‐glycine (FG)‐containing peptide, part of the nuclear pore complex involved in controlling the traffic between cytoplasm and cell nucleus. It is shown that the 23Na NMR together with the 17O NMR provide a detailed picture of the sodium ion and water mobility within the interior of the FG peptide hydrogel. As another example, we study phase separation in water‐triethylamine (TEA) mixture and provide evidence for the presence of multiple microscopic environments within the TEA‐rich phase. Our results highlight the potentials of 23Na NMR in combination with 17O NMR in studying biological phase separation, in particular with regards to the molecular properties of biomolecular condensates and their regulation through various physico‐ and biochemical factors.

Conformational Dynamics of Intrinsically Disordered Proteins Regulate Biomolecular Condensate Chemistry

“…Rezaei-Ghaleh and co-workers proposed 17 O NMR R 1 relaxation spectroscopy 170 and more recently 23 Na NMR chemical shift, R 1 relaxation, and pulse field gradient diffusion spectroscopy 171 to probe solvent properties in phase-separating systems. Notably, in water–glycerol mixtures of different viscosities, relative 17 O and 23 Na R 1 rates (ratio of R 1 rate measured in water–glycerol mixtures to the rate measured in water) scaled differently with the viscosity of the mixture.…”

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

“…Finally, signals from probe molecules may be recorded with a few scans, allowing observations of kinetics at significantly faster timescales. Other small molecules, such as sodium ions in 23 Na NMR or ammonium ions in 14 N NMR 37 , 38 , have also been shown to have potential uses as NMR probes for studying biological condensates or phase separation.…”

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

“…Finally, signals from probe molecules may be recorded with a few scans, allowing observations of kinetics at signi cantly faster timescales. Other small molecules, such as sodium ions in 23 Na NMR or ammonium ions in 14 N NMR, 40,41 have also been shown to have potential uses as NMR probes for studying biological condensates or phase separation.…”