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 (23) Na NMR as a proxy to report the internal fluidity of biomolecular condensed phases. After establishing the temperature and viscosity dependence of (23) Na NMR relaxation rates and translational diffusion coefficient, we demonstrate that (23) Na 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 (23) Na 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 (23) Na NMR together with the (17) O 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 (23) Na NMR in combination with (17) O 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.