Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation

Abstract

Membrane encapsulation is frequently used by the cell to sequester biomolecules and compartmentalize their function. Cells also concentrate molecules into phase-separated protein or protein/nucleic acid "membraneless organelles" that regulate a host of biochemical processes. Here, we use solution NMR spectroscopy to study phase-separated droplets formed from the intrinsically disordered N-terminal 236 residues of the germ-granule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, [Formula: see text], diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing [Formula: see text] to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of [Formula: see text] have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. Observation of a network of interchain interactions, as established by NOE spectroscopy, shows the importance of Phe and Arg interactions in driving the phase separation of Ddx4, while the salt dependence of both low- and high-concentration regions of phase diagrams establishes an important role for electrostatic interactions. The diffusion of a series of small probes and the compact but disordered 4E binding protein 2 (4E-BP2) protein in [Formula: see text] are explained by an excluded volume effect, similar to that found for globular protein solvents. No changes in structural propensities of 4E-BP2 dissolved in [Formula: see text] are observed, while changes to DNA and RNA molecules have been reported, highlighting the diverse roles that proteinaceous solvents play in dictating the properties of dissolved solutes.

Protein Phase Separation: A New Phase in Cell Biology

“…The range of sequence biases associated with IDRs that mediate phase separation indicates that there may be a range of underlying driving forces. These likely include electrostatic, dipole–dipole, pi–pi, cation–pi, hydrophobic, and hydrogen bonding interactions [9,49,50,52–56] (Figure 2A,B). Indeed, mutational studies have demonstrated that phase separation of different LCDs can be prevented by interfering with a variety of residue types [9,10,14,16,57].…”

“…A different mechanism is suggested by studies of liquid droplets formed by full-length FUS and hnRNPA1, or their LCDs [14,16]. In such liquid droplets, the LCDs seem to retain their tendency to be disordered, which is similar to their monomeric state [10,56]. However, for many droplets formed by low-complexity IDRs, especially in the case of constructs that contain the full-length protein, an eventual maturation into fibrillar solid aggregates occurs.…”

Heterochromatin Protein HP1α Gelation Dynamics Revealed by Solid‐State NMR Spectroscopy

“…[18] Biophysical treatments of LLPS proteins,s uch as HP1a, suggest that the compaction state,i nteraction strength, and multivalencyo ft he biomolecule can shape the collective material properties. [22][23][24] Solution NMR spectroscopy,h owever,cannot capture the molecular picture of biomolecules in the gel state where the increased viscosity and cross-linking interactions reduce molecular tumbling and cause severe linebroadening in the NMR spectra. [22] Some details have emerged from solution nuclear magnetic resonance (NMR) spectroscopy experiments of the dense liquid phase that point to the importance of intrinsically disordered protein regions and their transient interactions with other LLPS components.…”

Physical Chemistry of Cellular Liquid‐Phase Separation

“…Ddx4’s sequence is arranged in 8–10 residue blocks of alternating charge density, and this patterning is intrinsic to Ddx4’s behavior: a scrambled construct of identical composition was unable to phase separate under physiological conditions, despite accumulating to high concentrations in cells . In addition, arginine methylation and phenylalanine mutation or disruption both abrogated droplet formation (Figure ) …”