Programmable 2- and 3-Dimensional Protein Assemblies

无论是用于构建复杂的生物设备，如酶，还是用于构建具有高级物理特性的复杂材料，如骨骼肌，蛋白质在整个进化过程中一直是自然界的首要构建块。然而，实验室科学家控制蛋白质自组装或将其用作合成构件以产生新功能的能力受到严重限制。拟议研究计划的总体目标是通过开发基于金属配位化学和DNA纳米技术的新蛋白质设计工具来克服这一限制。在一个项目目标中，PI的团队将构建复杂的笼状蛋白质结构，最终可用于以刺激依赖的方式封装和释放分子货物。在第二个项目目标中，他们将利用DNA自组装的可编程性来生成多组分蛋白质结构，并阐明蛋白质-DNA相互作用的复杂能量景观。就更广泛的影响而言，拟议的努力不仅将提供对生物自组装过程的基本见解，而且还将提供对新的基于蛋白质的功能和材料的访问，这些功能和材料在生物/纳米技术和基础科学中具有潜在的应用（例如，分离、催化、传递、传感、基于X射线或电子衍射的结构测定）。由于其高度跨学科的性质，该项目将为研究生，本科生和高中研究人员提供广阔的培训基地，并帮助他们在职业生涯后期解决复杂的科学问题。作为一项新的教育活动，PI的实验室将开始举办为圣地亚哥地区高中的初级预备役军官训练团设计的STEM活动。这些活动将共同提高学生对蛋白质作为生物分子及其自组装/结晶的理解，以及它们作为新型催化剂和材料的构建块的新用途。重要的是，学生将接触到正在研究实验室发生的日常活动。虽然自然进化在数十亿年的时间里产生了卓越的基于蛋白质的机器和材料，但它只探索了可用蛋白质折叠实现的设计/自组装空间的极小部分。因此，非常希望能够使用选择的构建块从头开始制造有序的蛋白质组装体，这将大大拓宽天然存在的蛋白质组装体和生物启发材料的结构和功能范围。PI先前的研究结果表明，超分子/无机/聚合物化学和蛋白质工程的工具和原理可以以新的方式结合起来，以解决蛋白质自组装中的突出问题，并创造新型生物材料。在拟议的研究中，将在两个目标下扩大这些努力。在目标1下，PI的团队将通过使用非天然金属螯合功能来扩展他们的无机化学工具包，并将它们与计算设计联合收割机相结合，以获得以前无法实现的复杂的，刺激响应的多面体结构。目标2将利用DNA自组装的可编程性来产生异聚/多组分蛋白质结构，并更好地理解蛋白质-DNA共组装的复杂能量景观。目标将利用两种蛋白质构建块（细胞色素cb 562和Rop）以模块化方式访问大量寡聚和聚合蛋白质组装体，使用最先进的工具（例如，cryoEM，X射线衍射和散射），并检查它们的涌现功能（例如，该奖项反映了NSF的法定使命，并通过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，被认为值得支持。

Nontechnical SummaryWhether for constructing complex biological devices like enzymes or for building sophisticated materials with advanced physical properties like skeletal muscles, proteins have been nature’s premier building blocks throughout the course of evolution. Yet, the ability of laboratory scientists to control the self-assembly of proteins or to use them as synthetic building blocks to generate new functions has been severely limited. The overarching goal of the proposed research program is to overcome this limitation by developing new protein design tools based on metal coordination chemistry and DNA nanotechnology. In one project goal, the PI’s group will construct complex, cage-like protein architectures which can ultimately be used to encapsulate and release molecular cargo in a stimuli-dependent fashion. In a second project goal, they will exploit the programmability of DNA self-assembly to generate multicomponent protein architectures and elucidate the complex energy landscape of protein-DNA interactions. In terms of the broader impacts, the proposed effort will not only provide fundamental insights into biological self-assembly processes, but also lend access to novel protein-based functionalities and materials with potential applications in bio/nano-technology and basic sciences (e.g., separations, catalysis, delivery, sensing, X-ray or electron-diffraction-based structure determination). Due to its highly interdisciplinary nature, this project will provide an expansive training ground for graduate, undergraduate, and high school researchers, and help them tackle complex scientific problems later in their careers. As a new educational activity, the PI’s lab will start hosting STEM activities designed for Junior Reserve Officers’ Training Corps from San Diego-area high schools. These activities collectively will enhance the students’ understanding of proteins as biomolecules and their self-assembly/crystallization as well as their novel uses as building blocks for novel catalysts and materials. Importantly, the students will be exposed to daily activities that are taking place in a research laboratory. Technical SummaryWhile natural evolution has produced remarkable protein-based machines and materials over the course of billions of years, it has only explored an infinitesimally small fraction of the design/self-assembly space that could be achieved with the available protein folds. Therefore, it would be highly desirable to have the ability to craft ordered protein assemblies from scratch using building blocks of choice, which would greatly broaden the structural and functional scope of naturally existing protein assemblies and bioinspired materials. Previous findings from the PI’s indicate that the tools and principles of supramolecular/inorganic/polymer chemistry and protein engineering can be combined in new ways to address outstanding issues in protein self-assembly and to create novel biomaterials. In the proposed research, these efforts will be expanded under two Objectives. Under Objective 1, the PI’s group will expand their inorganic chemical toolkit through the use of unnatural metal chelating functionalities and combine them with computational design to access complex, stimuli-responsive polyhedral architectures that were previously out of reach. Objective 2 will exploit the programmability of DNA self-assembly to generate heteromeric/multicomponent protein architectures and to better understand the complex energy landscape of protein-DNA co-assembly. The Objectives will take advantage of two protein building blocks (cytochrome cb562 and Rop) to access a multitude of oligomeric and polymeric protein assemblies in a modular fashion, probe their structures and structural dynamics using state-of-the-art tools (e.g., cryoEM, X-ray diffraction and scattering), and examine their emergent functions (e.g., nucleic acid encapsulation).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.