Investigating the physicochemical properties of coacervates formed by intrinsically disordered proteins.

在过去的10年里，对由本质上无序的蛋白质形成的液体冷凝物的研究呈爆炸式增长。这些蛋白质凝聚物或液滴通常表现出生化活性，如RNA封存。DDX4是一种RNA加工蛋白质，具有固有的无序结构域，在足够高的蛋白质浓度下，将在水溶液中形成富含蛋白质的液滴。这些液滴在体外可以作为生物分子过滤器，优先吸收较短的调节RNA和融化较短的DNA双链。目前这种生化活性的机制尚不清楚。已经有一些工作将蛋白质序列与液滴的形成和活性联系起来，但这仍处于起步阶段。液滴界面也很少或根本没有表征，但它们的结构决定了它们对辅助因素的渗透性、润湿行为和液滴生长速度。这个项目将建立在之前关于Ddx4的研究的基础上，TJ Nott博士发表了三篇关于这种蛋白质的高影响力的论文。与LAF1和FUS等其他凝析油形成蛋白不同，DDX4可以可靠地形成不会出现明显老化现象的液滴。定制蛋白质设计是合成生物学的首要目标。通过揭示控制Ddx4行为的机制，我将能够合理设计具有可调特性的凝析形成蛋白。研究对象1.Ddx4液滴与寡核苷酸的选择性相互作用众所周知，Ddx4液滴将以一种对其二级结构具有选择性的方式吸收寡核苷酸。将研究寡核苷酸在吸收时的构象变化。我将记录在5‘和3’位置用荧光团标记的寡核苷酸在吸收到蛋白质液滴时Forster共振能量转移(FRET)信号的变化。2.蛋白质液滴的界面特性与Dirk Aart教授合作，将使用胶体科学的方法来研究ddx4液滴的界面特性。其目标是从软凝聚物质领域找到产生关于液滴界面的有形的、生物学上相关的结论的方法和模型。液滴成核的机理将通过研究液滴形成后不久的空间分布来研究，并在一系列参数范围内进行探索。通过研究液滴在一系列功能化表面上的成核和润湿，将开发一种表面性质的测试方法。我希望使用相差显微镜来拍摄没有任何荧光标记的液滴。3.将液滴功能与序列联系起来将确定影响蛋白质-DNA相互作用和Ddx4液滴界面性质的序列特征。通过克隆和定点突变，我将产生一系列突变体Ddx4‘S。感兴趣的修改如下。-已知导致DDX4蛋白缩合的残基的点突变。这些修饰的DDX4‘S将受到与点1和点2相同的方法。我将调查DDX4的选择性寡核苷酸吸收和界面属性耦合的程度，因此这两个属性是否可以独立调节。我希望引入一种能够在体外与寡核苷酸相互作用中编码选择性的液滴形成蛋白。由于这些液滴经常表现出生化活性，如RNA隔离，它们将在生物医学应用的合成组织设计中得到应用。DPhil项目属于EPSRC的“生物物理和软物质物理”和“合成生物学”研究领域。伴随着研究主题，我还将在OxSyBio花两个月的时间进行工业安置，他们正在开发3D细胞打印平台，并正在研究由活性液滴形成的合成组织。

In the last 10 years there has been an explosion of research into liquid condensates formed by intrinsically disordered proteins. These protein condensates, or droplets, often display biochemical activity such as RNA sequestration. Ddx4 is an RNA processing protein with an intrinsically disordered domain that, at high enough protein concentrations, will form protein rich droplets in aqueous solution. These droplets can act as biomolecular filters in vitro, preferentially absorbing shorter regulatory RNA's and melting short DNA duplexes. The mechanism of this biochemical activity is currently unknown.There has been some work relating protein sequence to the formation and activity of liquid droplets, but this is still in its infancy. There is also little to no characterisation of droplet interfaces, yet their structure determines their permeability to cofactors, wetting behaviour and rate of droplet growth. This project will build on previous research on Ddx4, Dr TJ Nott has three high impact papers published on this protein. Ddx4 reliably forms droplets that do not show appreciable ageing phenomena, unlike other condensate forming proteins such as LAF1 and FUS. Bespoke protein design is an overarching goal of synthetic biology. By uncovering the mechanisms that govern the behaviour of Ddx4 I will enable to rational design of a condensate forming protein with tuneable properties. Research Objectives1. Selective interactions of Ddx4 droplets with oligonucleotidesIt is known that Ddx4 droplets will absorb oligonucleotides in a manner that is selective for their secondary structure. Conformational changes of oligonucleotides upon absorption will be investigated. I will record changes in the Forster resonance energy transfer (FRET) signal for oligonucleotides labelled at the 5' and 3' positions with fluorophores upon absorption into protein droplets. 2. Interfacial properties of protein dropletsIn collaboration with Prof Dirk Aarts, methods from colloid science will be used to study the interfacial properties of ddx4 droplets. The goal is to find methods and models from the field of soft condensed matter that yield tangible, biologically relevant conclusions about droplet interfaces. The mechanism of droplet nucleation will be studied by investigating the spatial distribution of droplets shortly after their formation, exploring this over a range of parameters. An assay for surface properties will be developed by studying the nucleation and wetting of droplets on a range of functionalised surfaces. I hope to use phase contrast microscopy to image droplets that lack any fluorescent tag. 3. Relating droplet functionality to sequenceThe sequence features that effect protein-DNA interactions and interfacial properties of Ddx4 droplets will be determined. Through cloning and site directed mutagenesis, I will generate a range of mutant Ddx4's. The modifications of interest are as follows. - Point mutations of residues known to contribute to the condensation.- Elongation of the Ddx4 protein.These modified Ddx4's will then be subjected to the same methods as in points 1 and 2. I will investigate the degree to which the selective oligonucleotide absorption and interfacial properties of Ddx4 are coupled, and therefore whether these two properties are independently tuneable. I hope to introduce a droplet forming protein with the ability to encode selectivity in its in vitro interactions with olignucleotides. Because these droplets often display biochemical activity, such as RNA sequestration, they will have applications in the design of synthetic tissues for biomedical applications. The DPhil project falls within the EPSRC 'Biophysics and Soft Matter Physics' and 'Synthetic Biology' research areas. Accompanying the research themes, I will also spend 2 months on an industrial placement at OxSyBio who are developing both a 3D cell printing platform and are studying synthetic tissues formed from active droplets.