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

研究由本质上无序的蛋白质形成的凝聚层的物理化学性质。

• 批准号: 1934917
• 金额: --
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1934917
• 关键词: Investigating; physicochemical; properties; coacervates; formed

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.

在过去的十年里，对由内在无序的蛋白质形成的液体凝聚体的研究出现了爆炸式的增长。这些蛋白质凝聚物或液滴通常表现出生化活性，如RNA隔离。Ddx4是一种具有内在无序结构域的RNA加工蛋白，在足够高的蛋白质浓度下，会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和融化短的DNA双链。这种生物化学活性的机制目前尚不清楚。已经有一些工作将蛋白质序列与液滴的形成和活动联系起来，但这仍处于起步阶段。液滴界面也几乎没有表征，但它们的结构决定了它们对辅因子的渗透性、润湿行为和液滴生长速度。该项目将建立在之前对Ddx4的研究基础上，TJ Nott博士已经发表了三篇关于这种蛋白质的高影响力论文。Ddx4可靠地形成液滴，不显示明显的老化现象，不像其他凝析形成蛋白质，如LAF1和FUS。定制蛋白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聚形成蛋白。研究Objectives1。Ddx4液滴与寡核苷酸的选择性相互作用众所周知，Ddx4液滴会以一种对其二级结构有选择性的方式吸收寡核苷酸。寡核苷酸在吸收后的构象变化将被研究。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滴时的福斯特共振能量转移（FRET）信号的变化。2. 与Dirk Aarts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。目标是找到软凝聚态物质领域的方法和模型，从而产生关于液滴界面的有形的、生物学相关的结论。液滴成核的机制将通过研究液滴形成后不久的空间分布来研究，并在一系列参数上进行探索。通过研究液滴在一系列功能化表面上的成核和润湿，将开发一种表面特性测定方法。我希望使用相衬显微镜来成像没有任何荧光标记的液滴。3. 将液滴功能与序列联系起来影响蛋白- dna相互作用和Ddx4液滴界面特性的序列特征将被确定。通过克隆和定点诱变，我将产生一系列Ddx4突变体。利息的修改如下。-已知有助于凝结的残基的点突变。- Ddx4蛋白的延伸。然后，这些修改后的Ddx4将受到与第1点和第2点相同的方法。我将研究Ddx4的选择性寡核苷酸吸收和界面特性耦合的程度，以及这两种特性是否可以独立调节。我希望介绍一种液滴形成蛋白，它在体外与寡核苷酸的相互作用中具有编码选择性的能力。由于这些液滴经常显示生化活性，例如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。博士项目属于EPSRC的“生物物理学和软物质物理学”和“合成生物学”研究领域。伴随着研究主题，我还将花2个月的时间在OxSyBio进行工业实习，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。