Protein Crystallization Programmed with DNA

第一部分： 蛋白质是天然的微小分子机器-通常比人类头发的宽度小10，000倍-在生物学中起着至关重要的作用。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，它们的微小尺寸使它们难以描述和理解。确定蛋白质结构和功能的一种有效方法是蛋白质晶体学，这是一种X射线与高度有序的蛋白质组装体（称为单晶）相互作用的技术。不幸的是，获得蛋白质单晶是这一过程中的一个主要瓶颈，因为蛋白质可以以多种方式相互作用，从而阻止它们形成高度有序的组装体。该项目旨在克服这一挑战，使用DNA -生命的遗传密码-作为蓝图来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，这些单晶不仅可以让我们深入了解蛋白质的微小世界，还可以作为新的合成材料，作为可持续催化剂或能量转换材料。利用DNA来控制蛋白质的相互作用和排列，将使晶体能够通过设计组装起来。这项工作将有助于将蛋白质晶体学从偶然性实验转变为有目的的实验，以解决能源，可持续性和医学方面的紧迫社会需求。处于职业生涯各个阶段的研究人员（从本科生到博士后研究人员）将受益于本项目提供的培训，并将通过出版物、演讲以及通过协同外联活动与来自代表性不足和边缘化群体的学生接触来传播他们的知识和技能。 技术概述蛋白质单晶为产生生命基础结构的大分子提供了有价值的埃级分辨率和结构洞察力，并代表了一类具有合作性质和协调功能的有前途的生物材料。该项目旨在了解驱动结晶的相互作用，并发现一种方法来破坏，重新编程和重新定义这些相互作用，使用DNA的可编程性。这一挑战将从四个互补的角度来处理，每一个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;研究对称性和化合价如何影响结晶;定义晶体内的特定蛋白质界面;操纵柔性蛋白质的构象，以便它们可以被控制并最终被利用。这些发现将导致设计原则，将允许人们利用DNA的许多不同属性，包括其特异性杂交，可调长度，固有的灵活性和可定制的相互作用强度，以编程蛋白质的组装。因此，实现这些目标不仅将使具有挑战性的蛋白质适合晶体学分析，而且重要的是，开辟了一类新的可定制的，可编程的晶体材料，可以利用蛋白质的内在功能。该项目将产生关于如何控制DNA-DNA和蛋白质-蛋白质相互作用之间的相互作用的新的基础知识，从而使研究人员能够利用工具设计蛋白质结晶的结构结果，以创造新的功能性生物材料。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.