Reprogramming materials properties of synapsin self-coacervates via phosphorylation code

非技术总结在寻找细胞内的结构以启发下一代新材料方面，最有趣的方面可能不是它们的整体结构特性，而是细胞对这些结构进行重新编程的能力。就像重新编程软件允许它执行不同的任务一样，重新编程材料允许它们在生产后具有新的功能。这一点在精选的蛋白质中尤其明显，这些蛋白质分成富含蛋白质和稀释蛋白质的液体相，就像油加到水中会分离成富油的液体一样。这种相分离已经被证明是由磷酸化控制的，也就是在蛋白质上增加一个带电的磷酸基团。并不是所有的磷酸化都是一样的；蛋白质中某些位置的磷酸化将消除相分离的能力，而其他位置仍然可以控制分子进出富含蛋白质的液体。然而，由于不能同时评估细胞中富含蛋白质的液体的磷酸化序列(或“磷酸化密码”)和相应的材料性质，因此很难设计出具有类似重编程能力的合成材料。本项目试图克服这一障碍，通过使用新的生物化学工程技术来制造具有特定磷酸化密码的蛋白质，并随后测量这些相分离蛋白质的材料性质。在这样做的过程中，将创建一个框架，以设计具有类似重新编程能力的其他材料，并可能产生一类新的仿生材料，用于治疗和消费。除此之外，鉴于多学科的科学和工程要求，该项目不仅将为下一代跨学科科学家提供培训机会，还将通过为大洛杉矶地区当地社区大学的学生提供研究机会来扩大这一渠道。技术摘要内在无序蛋白质(IDPs)在溶液中以生物聚合物的形式存在，并可经历相分离形成富含蛋白质的液滴，类似于聚合物凝聚。这些亚细胞结构具有独特的用途，从细胞器隔离到在细胞内潜在地充当生物反应器。然而，生物学已经建立了一层额外的复杂性；内源性DP可以通过翻译后修饰来改变材料属性，从而实现一系列对于折叠良好的蛋白质来说不切实际的动态性质。一种特殊的修饰，磷酸化(或添加二价带电的磷酸基团)已被证明独立控制突触素的相行为和生物大分子运输特性，突触素是一种内源性DP，在神经元内形成富含突触素的液滴。尽管对这些特性的控制是生物材料研究的一个诱人的目标，但破译可能发生在多个不同位置的修饰的“磷酸化密码”，并将这些密码与其产生的生物功能关联起来，仍然是一个具有挑战性的障碍。为了克服这一障碍，来自聚合物科学和物理化学的见解将被用来针对最有可能改变材料性质的特定磷酸化代码。然后，无细胞蛋白质合成策略将被用来表达和纯化具有特定磷酸化密码的突触素，并测量其合成的生物材料的性质。最终，这些数据将成为设计框架的基础，该框架具有对其他基于IDP的生物材料甚至合成聚合物系统进行重新编程的解释和预测能力。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

NON-TECHNICAL SUMMARYPerhaps the most intriguing aspect in looking to structures within the cell to inspire the next generation of novel materials is not their bulk structural properties but the cellular ability to “reprogram” these structures. Much as reprogramming software allows it to perform different tasks, reprogramming materials allows them to take on novel functions after production. This is especially evident in select proteins that phase separate into protein-rich and protein-dilute liquid phases, much as oil added to water will separate into an oil-rich liquid phase. This phase separation has been shown to be controlled by phosphorylation, or the addition of a charged phosphate group to the protein. Not all phosphorylations are created equal; phosphorylation at certain sites within the protein will eliminate the ability to phase separate while others still can control the transport of molecules in-and-out of the protein-rich liquid phase. However, the inability to simultaneously assess the sequence of phosphorylation (or “phosphorylation code”) and the corresponding materials properties of the protein-rich liquid phase in the cell has made it difficult to design synthetic materials that offer similar reprogramming capability. This project seeks to overcome this obstacle by using novel biochemical engineering techniques to manufacture proteins with specific phosphorylation codes and subsequently measure the materials properties of these phase separating-proteins. In doing so, a framework will be created to design other materials with similar reprogramming capabilities and potentially generate a new class of biomimetic materials for therapeutic and consumer use. Beyond that, given the multidisciplinary scientific and engineering requirements, this project will not only provide training opportunities for the next generation of interdisciplinary scientists but seek to expand that pipeline by providing research opportunities for students at local community colleges in the greater Los Angeles area. TECHNICAL SUMMARYIntrinsically disordered proteins (IDPs) persist as biopolymers in solution and can undergo phase separation into protein-rich liquid droplets, akin to polymer coacervation. These subcellular structures serve unique purposes, ranging from organelle sequestration to potentially behaving as bio-reactors within the cell. However, biology has built in an additional layer of sophistication; IDPs can be “reprogrammed” via post-translational modifications to alter the materials properties, enabling a range of dynamic properties that would be impractical with well-folded proteins.One particular modification, phosphorylation (or the addition of a divalently-charged phosphate group), has been shown to independently control the phase behavior and biomacromolecule transport properties of synapsin, an IDP that forms synapsin-rich liquid droplets within the neuron. While control of these properties is an inviting target for biomaterials investigation, deciphering the “phosphorylation code” of modifications that can occur at multiple, distinct sites and correlating these codes to their resultant biological function remains a challenging obstacle. To overcome this barrier, insights from polymer science and physical chemistry will be used to target specific phosphorylation codes most likely to alter materials properties. Then, cell-free protein synthesis strategies will be used to express and purify synapsin with specific phosphorylation codes and measure their resultant biomaterials properties. Ultimately, this data will form the basis of a design framework that has explanatory and predictive power for reprogramming other IDP-based biomaterials and even synthetic polymer systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.