5 years ago

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation [Biophysics and Computational Biology]

Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation [Biophysics and Computational Biology]
Lewis E. Kay, Patrick J. Farber, Jacob P. Brady, Hue Sun Chan, Timothy J. Nott, Rui Huang, Julie D. Forman-Kay, Andrew J. Baldwin, Yi-Hsuan Lin, Ashok Sekhar, Alaji Bah

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, Ddx4condDdx4cond, diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4condDdx4cond to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4condDdx4cond 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 Ddx4condDdx4cond 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 Ddx4condDdx4cond 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.

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