Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation
Structural and hydrodynamic properties of an intrinsically disordered region of a germ cell-specific protein on phase separation
复制标题
DOI：
10.1073/pnas.1706197114
复制
发表时间：
2017-09-26
影响因子：
11.1
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Brady, Jacob P.;Farber, Patrick J.;Kay, Lewis E.
细胞经常使用膜封装来隔离生物分子并分裂其功能。细胞还将分离分子集中在调节大量生化过程的相位分离蛋白或蛋白质/核酸“膜无细胞器”中。在这里，我们使用溶液NMR光谱法研究由固有无序的N末端236残基形成的相分开的液滴。我们表明，分离ddx4，ddx4（cond）的集中阶段中的蛋白质扩散为溶于水中的600 nm流体动力半径的粒子。然而，NMR光谱显示出具有化学位移的急剧共鸣，表明DDX4（COND）本质上是无序的。自旋松弛测量表明，DDX4（COND）的骨干酰胺具有明显的迁移率，解释了为什么观察到高分辨率光谱，但与等效浓缩的非相分离对照相比，运动降低了。通过NOE光谱证明，观察互链相互作用网络的观察表明，PHE和ARG相互作用在驱动DDX4的相位分离中的重要性，而相图的低浓度和高浓度区域的盐依赖性确立了重要的作用用于静电相互作用。一系列小探针和DDX4（COND）中的紧凑但无序的4E结合蛋白2（4E-BP2）蛋白的扩散通过排除的体积效应来解释，类似于球状蛋白溶剂发现的体积效应。未观察到溶解在DDX4（COND）的4E-BP2的结构倾向的变化，而据报道了DNA和RNA分子的变化，强调了蛋白质溶剂在决定溶解溶质的性质中起着蛋白质溶剂在决定性质中起作用的多种作用。
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 germgranule protein Ddx4. We show that the protein within the concentrated phase of phase-separated Ddx4, Ddx4(cond), diffuses as a particle of 600-nm hydrodynamic radius dissolved in water. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4(cond) to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4(cond) 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 Ddx4(cond) 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 Ddx4(cond) 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.