Protein Crystallization Programmed with DNA

第一部分：非技术的SUMMARY蛋白质是一种天然的微小分子机器--通常比人类头发的宽度小一万倍--在生物学中扮演着至关重要的角色。自从近200年前发现它们以来，了解它们的结构和功能使科学家能够了解支撑生命的过程，并解决人类面临的科学问题。然而，它们的微小尺寸使得它们的特征和理解变得极其困难。确定蛋白质结构和功能的一种有效方法是蛋白质结晶学，这是一种X射线与蛋白质高度有序的组装相互作用的技术，称为单晶。不幸的是，获得蛋白质单晶是这一过程中的一个主要瓶颈，因为蛋白质可以通过许多方式相互作用，阻止它们形成高度有序的组装。该项目旨在克服这一挑战，使用DNA--生命的遗传密码--作为蓝图，定义蛋白质之间的相互作用，从而控制它们如何排列成单晶。重要的是，这些单晶不仅将提供对蛋白质微小世界的洞察，而且将本身作为新的合成材料发挥作用，作为可持续的催化剂或能量转换材料。使用DNA来控制蛋白质的相互作用和排列，将允许晶体按设计组装。这项工作将有助于将蛋白质结晶学从一种偶然的实验转变为一种有目的的实验，以解决能源、可持续发展和医学方面迫切的社会需求。处于职业生涯不同阶段的研究人员(从本科生到博士后研究人员)将从该项目提供的培训中受益，并将在出版物、演示文稿中传播他们的知识和技能，并通过协同外联活动与来自典型代表不足和边缘化群体的学生接触。第2部分：技术总结蛋白质单晶提供有价值的、埃级别的分辨率和对构成生命基础设施的大分子的结构洞察，并代表一类具有合作特性和协调功能的有前途的生物材料。这个项目试图了解驱动结晶的相互作用，并发现一种利用DNA的可编程性来中断、重新编程和重新定义这些相互作用的方法。这一挑战将从四个互补的角度来解决，每个角度都将产生对蛋白质结晶的宝贵的基本见解：增加DNA在蛋白质-DNA晶体中的作用；研究对称性和价态如何影响结晶；确定晶体中特定的蛋白质界面；以及操纵灵活的蛋白质的构象，以便能够控制并最终利用它们。这些发现将产生设计原则，使人们能够利用DNA的许多不同属性，包括其特定的杂交、可调的长度、固有的灵活性和可定制的相互作用强度，来编程蛋白质的组装。因此，实现这些目标不仅将使具有挑战性的蛋白质易于进行结晶学分析，而且重要的是，还将开辟一类新的可定制的、可编程的晶体材料，可以利用蛋白质的内在功能。这个项目将产生关于如何控制DNA-DNA和蛋白质-蛋白质相互作用的新的基础知识，从而使研究人员能够利用工具来设计蛋白质结晶的结构结果，以创造新的功能生物材料。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。

PART 1: NON-TECHNICAL SUMMARYProteins are natural, tiny molecular machines – often 10,000 times smaller than the width of a human hair – that play crucial roles in biology. Since their discovery almost 200 years ago, understanding their structures and functions has enabled scientists to learn about the processes that underpin life and solve scientific problems facing humanity. However, their tiny size makes them incredibly difficult to characterize and understand. One powerful way to determine the structures and functions of proteins is protein crystallography, a technique where X-rays interact with a highly ordered assembly of proteins, known as a single crystal. Unfortunately, obtaining protein single crystals represents a major bottleneck in this process because proteins can interact with each other in many ways that prevent them from forming highly ordered assemblies. This project aims to overcome this challenge using DNA – the genetic code of life – as a blueprint to define the interactions between proteins and thus control how they arrange into single crystals. Importantly, these single crystals will not only provide insight into the tiny world of proteins but will function as new, synthetic materials in their own right, useful as sustainable catalysts or energy conversion materials. Using DNA to control protein interactions and arrangement will allow crystals to be assembled by design. This work will help transform protein crystallography from an experiment of chance to an experiment of purpose to solve pressing societal needs in energy, sustainability, and medicine. Researchers at various stages of their careers (from undergraduate students to postdoctoral researchers) will benefit from the training provided by this project, and will disseminate their knowledge and skills in publications, presentations, and by engagement with students from typically underrepresented and marginalized groups through synergistic outreach activities.PART 2: TECHNICAL SUMMARYProtein single crystals provide valuable, angstrom-level resolution and structural insight into the macromolecules that engender the infrastructure of life and represent a promising class of biomaterials with cooperative properties and concerted functions. This project seeks to understand the interactions that drive crystallization and discover a means to disrupt, reprogram, and redefine those interactions, using the programmability of DNA. This challenge will be approached from four complementary perspectives, each of which will yield valuable fundamental insight into protein crystallization: increasing the role of DNA in protein-DNA crystals; investigating how symmetry and valency affect crystallization; defining specific protein interfaces within crystals; and manipulating the conformations of flexible proteins so that they can be controlled and, ultimately, harnessed. These findings will result in design principles that will allow one to exploit the many distinct attributes of DNA, including its specific hybridization, tunable length, inherent flexibility, and tailorable interaction strength, to program the assembly of proteins. Therefore, achieving these objectives will not only render challenging proteins amenable to crystallographic analysis but also, importantly, open a new class of tailorable, programmed crystalline materials that can harness the intrinsic functionality of proteins. This project will generate new fundamental knowledge of how to control the interplay between DNA-DNA and protein-protein interactions, thereby empowering researchers with tools to engineer the structural outcomes of protein crystallization towards the creation of novel functional biomaterials.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.