Deciphering the Sequence Code for Compositional Control of Phase Separated Biomolecular Condensates

项目摘要/摘要 通过液-液相分离(LLP)形成的生物分子凝聚体是 细胞生物化学。蛋白质LLP通常通过内在无序的蛋白质之间的接触或 较大蛋白质内的区域(IDPR)，通常与多价折叠相互作用结构域合作。 支持LLP的IDPR序列的空间仅被稀疏采样，这是由于低的- 当前使用的吞吐量技术。因此，描述IDPR之间的联系的一般框架 初级序列、构象集合和LLP仍然难以捉摸。通过开发和应用小说 高通量方法，我提出的工作旨在打破经典生物化学的瓶颈。这就做 设计编码GFP标记的IDPR、不同氨基酸组成的~10,000个序列DNA文库 初级序列中的残基图案化。高通量基因将构建文库序列 合成和序列将单独表达的微流控液滴使用体外 转录/翻译。然后我将使用荧光激活的液滴分类来选择含有 支持LLP的序列，这将通过对排序的DNA进行高通量测序来识别 水滴。计算模拟和生化/生物物理表征将演示如何 在IDPR相分离中，初级序列和构象系综被联系在一起。进一步应用这一点 在此方法中，我将识别通过多价折叠结构域调节LLP的IDPR功能。最后，我将确定 IDPR功能控制凝析油的凝固速度，这是一个在疾病中加速的过程- 凝析油形成IDPR的相关突变体。拟议的工作将提供一套规则来管理 通过IDPR进行的相分离，得到了史无前例的经验数据集的支持。这项工作将是 在生物分子凝析油领域的先驱迈克尔·罗森博士的实验室里进行的 德克萨斯大学西南医学中心是一家领先的生物医学研究机构，拥有 广泛领域的设施和调查人员。作为这次研究培训计划的一部分，我将承担一项 一系列强有力的职业发展和培训活动，包括负责任的行为课程 研究、拨款撰写、实验室管理和教育技术。我也会有很多 有机会参加研讨会和会议，在那里我将能够在海报会议上展示我的工作 和演讲，以及在同行评议的期刊上发表我的作品的机会。我还计划发展一项教学 通过培训研究生、轮流学生和访问获得指导经验 本科生。总之，这里提出的研究培训计划将为我提供技能， 知识和经验是我实现成为一名独立调查员的长期目标所必需的。

Project Summary/Abstract Biomolecular condensates formed via liquid-liquid phase separation (LLPS) are important organizers of cellular biochemistry. Protein LLPS is often mediated by contacts between intrinsically disordered proteins or regions (IDPRs) within larger proteins, frequently in cooperation with multivalent folded interaction domains. The space of IDPR sequences that support LLPS has been only sparsely sampled, owing to the low- throughput techniques currently used. Thus, a general framework describing the connections between IDPR primary sequence, conformational ensemble, and LLPS remains elusive. By developing and applying novel high-throughput methods, the work I propose aims to break the bottlenecks of classical biochemistry. I will design a ~10,000-sequence DNA library encoding GFP-tagged IDPRs, varying amino acid composition and residue patterning in the primary sequence. Library sequences will be constructed by high-throughput gene synthesis and sequences will be individually expressed microfluidic droplets using in vitro transcription/translation. I will then use fluorescence-activated droplet sorting to select droplets bearing sequences that support LLPS, which will be identified by high-throughput sequencing of DNA from sorted droplets. Computational simulations and biochemical/biophysical characterization will demonstrate how primary sequence and conformation ensemble are connected in IDPR phase separation. Further applying this approach, I will identify IDPR features that modulate LLPS by multivalent folded domains. Finally, I will identify IDPR features that govern rates of condensate solidification, a process that is accelerated in disease- associated mutants of condensate-forming IDPRs. The proposed work will provide a set of rules governing phase separation by IDPRs, backed up by an empirical dataset of unprecedented scale. This work will be carried out in the lab of Dr. Michael Rosen, a pioneer in the field of biomolecular condensates, at the University of Texas Southwestern Medical Center, a leading biomedical research institute with excellent facilities and investigators in a wide array of fields. As part of this research training plan, I will be take on a robust series of career development and training activities, including coursework in the responsible conduct of research, grant writing, laboratory management, and educational techniques. I will also have numerous opportunities to attend seminars and conferences, where I will be able to present my work in poster sessions and talks, and opportunities to publish my work in peer-reviewed journals. I also plan to develop a teaching portfolio and gain mentorship experience by training graduate students, rotation students, and visiting undergraduates. In summary, the research training plan proposed here will provide me with the skills, knowledge, and experience necessary to fulfill my long-term goal of becoming an independent investigator.