CAREER: Post-translationally Lipidated Biopolymers As Multiphasic All-Aqueous Emulsions

该奖项全部或部分由2021年美国救援计划法案（公法117-2）资助。第1部分：非技术概述乳液是两种不混溶液体的悬浮液，在许多行业中广泛用于各种应用。这个CAREER项目的研究目标是使用受自然细胞生物学启发的工艺合成基于生物材料的水包水乳液。细胞通过利用其细胞内蛋白质的相分离来产生这样的乳液，并且已经开发出通过使用不同的化学反应调色板修饰相分离蛋白质来定制这些乳液的性质的能力。我们对这些改性如何稳定或调整水-水界面的理解仍然不完整，这增加了我们对传统油基乳液的依赖。为了应对这一挑战并推进生物材料设计领域，本研究使用遗传和化学操作的组合来合成和研究具有可定制修饰模式的相分离蛋白质。将表征所得乳液的尺寸和稳定性，并将其与改性模式相关联。从这些研究中获得的知识将用于创建水包水乳液，可以精确控制以具有用户定义的特性。由于它们的生物相容性，这种新型水包水乳液在食品加工、化妆品、生物传感和药物递送中具有替代传统油基乳液的巨大潜力。该项目将为材料科学界提供创新且易于使用的平台，用于可扩展的生物材料合成，并有助于加快制造下一代结构复杂，功能可调的生物材料的能力，用于多种工业和医疗保健应用。这将加强美国。在全球生物经济中的领导地位。通过该项目支持的教育活动将培训下一代生物技术科学家和工程师。一个主要的教育活动是学生为织物和纺织品应用创造可生物降解和生态友好的材料。这项工作的应用可以解决高污染和环境破坏的纺织制造业的现行做法。这一教育活动将用于推广，以吸引和吸引高中学生参与科学和设计的合并。此外，该项目将为本科生和研究生实验室课程的重新设计提供信息，该课程将使学生参与现实世界的发现，并将在生物技术行业使用的监管框架中对学生进行培训。第二部分：技术概述本职业项目将利用翻译后修饰（PTM）的生物启用过程，以推进合理设计功能超过天然生物聚合物能力的混合生物材料的目标。该研究将解密相分离脂化蛋白（PLPs）的材料设计原理，以创建具有用户定义的形成，流体特性和内部结构的水包水乳液。该研究将调查总体假设，即蛋白质，脂质和脂化位点结构域之间的物理化学相互作用调节粘附和内聚相互作用的强度和半衰期，这些相互作用控制PLP缩合物的形成，粘弹性和分级组织。三个研究目标将阐明脂化PTM如何改变模型蛋白质缩合物的界面和流变学特性的分子和化学基础描述：（1）将PLP乳液的热力学稳定性与脂质和相分离蛋白的物理化学相关联，（2）确定脂化位点序列对两相PLP乳液的内聚相互作用和胶体性质的动力学的影响，和（3）确定调节多相PLP乳液的粘附性微观相互作用和宏观相容性的分子因素。实现这些目标的方法包括PLP的生物/半合成以创建具有系统变化的脂质、蛋白质和脂化位点的文库;使用光散射技术确定相边界;以及使用显微镜表征PLP液滴的动态材料性质和使用流变学表征相分离状态。多维核磁共振技术将用于研究PLPs的定义结构和动态元素，以实现具有可定制结构和材料特性的下一代冷凝物的合理设计。该教育计划将这些研究工作纳入多层次的方法，以（1）提高公众对生物材料和技术作为解决社会相关问题的认识，（2）招募来自非传统背景的代表性不足的少数民族进行生物材料研究，以及（3）加强本科生和研究生阶段的生物材料教育。该奖项反映了NSF的法定使命，并被认为值得通过以下方式支持：使用基金会的知识价值和更广泛的影响审查标准进行评估。

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2).PART 1: NON-TECHNICAL SUMMARY Emulsions are suspensions of two immiscible liquids, which are ubiquitously utilized across many industries for diverse applications. The research goal of this CAREER project is to synthesize biomaterial-based water-in-water emulsions using a process inspired by natural cell biology. Cells create such emulsions by leveraging phase separation of their intracellular proteins and have developed capabilities to tailor the properties of these emulsions by modifying the phase separating proteins using a diverse palette of chemical reactions. Our understanding of how these modifications stabilize or tailor the water-water interfaces remains incomplete, which increases our reliance on traditional oil-based emulsions. To address this challenge and advance the field of biomaterial design, this research uses a combination of genetic and chemical manipulation to synthesize and study phase-separating proteins with customizable modification patterns. The size and stability of resulting emulsions will be characterized and will be correlated to the modification patterns. The knowledge gained from these studies will be used to create water-in-water emulsions that can be precisely controlled to have user-defined characteristics. Due to their biocompatibility, such new water-in-water emulsions have great potential to replace traditional oil-based emulsions in food processing, cosmetics, biosensing, and delivery of pharmaceuticals. This project will provide the materials science community with innovative and easy-to-use platforms for scalable synthesis of biomaterials and help accelerate the ability to manufacture the next generation of structurally complex, functionally tunable biomaterials for multiple industrial and healthcare applications. This will strengthen the U.S.'s leadership in the global bioeconomy. Educational activities supported through this project will train the next generation of scientists and engineers in bio-enabled technologies. A major educational activity is the student creation of biodegradable and eco-friendly materials for fabric and textile applications. Applications of this work can address the highly polluting and environmentally damaging current practices in textile manufacturing. This educational activity will be used in outreach to attract and engage high school students to this merger of science and design. In addition, this project will inform the redesign of an undergraduate and graduate laboratory course that will involve students in real-world discovery and will train students in the regulatory framework used in the biotechnology industry. PART 2: TECHNICAL SUMMARY This CAREER project will exploit the bio-enabled process of post-translational modification (PTM) to advance the goal of rationally designing hybrid biomaterials with functionality that exceeds the capabilities of natural biopolymers. The study will decrypt the material-design principles of phase-separating lipidated proteins (PLPs) to create water-in-water emulsions with user-defined formation, fluid properties, and internal structure. The study will investigate the overarching hypothesis that the physicochemical interplay between the protein, lipid, and lipidation site domains regulates the strength and half-life of adhesive and cohesive interactions that control the formation, viscoelasticity, and hierarchical organization of PLP condensates. Three research objectives will elucidate the molecular and thermodynamically grounded descriptions of how lipidation PTM alters the interfacial and rheological characteristics of model protein condensates: (1) correlate the thermodynamic stability of PLP emulsions to the physicochemistry of lipid and phase-separating proteins, (2) ascertain the effect of lipidation site sequence on the dynamics of cohesive interactions and colloidal properties of biphasic PLP emulsions, and (3) determine the molecular factors that regulate the strength of adhesive microscopic interactions and macroscopic miscibility of multiphasic PLP emulsions. Methods to achieve these objectives include bio-/semi-synthesis of PLPs to create libraries with systematically varied lipid, protein, and lipidation sites; determination of phase boundaries using light scattering techniques; and characterization of dynamic material properties of PLP droplets using microscopy and phase-separated states using rheology. Multidimensional nuclear magnetic resonance techniques will be used to investigate the defining structural and dynamic elements of PLPs to enable the rational design of the next generation of condensates with tailorable structural and material properties. The education plan integrates these research efforts into a multitiered approach to (1) increase the public's awareness of bio-enabled materials and technologies as solutions to societally relevant problems, (2) recruit underrepresented minorities from nontraditional backgrounds to biomaterials research, and (3) enhance biomaterials education at the undergraduate and graduate levels.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.