Designed Expanded Co-Crystals for Guest Structure Determination

为了理解生物学和设计医疗干预措施，观察大型生物分子所采用的结构是必不可少的。确定这些结构的主要方法之一是诱导分子排列成晶体，然后收集这些晶体暴露在x射线束下时产生的衍射图样。这种传统工艺的故障率很高。因此，研究人员长期以来一直在寻求开发能够组织客体分子的支架晶体。这个项目将产生一类新的候选支架晶体。从技术上讲，这些新的晶体上层结构是共晶体，因为它们由蛋白质和DNA组成。为了确定这个家族中最有前途的材料，该团队将使用计算设计和全原子模拟来评估实验验证的候选材料。这些新的晶体材料将采用模块化设计，其中具有任何所需序列的“DNA支柱”暴露在开放的通道中，这些通道足够大，可以允许客体蛋白质的移动。研究小组将培养晶体，其中这些DNA柱具有被某些DNA结合蛋白特异性识别的序列。当这些客蛋白附着在它们的靶DNA上时，它们将加入晶格，并通过x射线衍射被观察到。这种方法绕过了随意的晶体生长过程，这是传统晶体学的基础。因此，由此产生的材料可能为科学家提供一种革命性的方法，使他们能够常规地、轻松地观察DNA和DNA结合蛋白之间复合物的原子细节，这些复合物驱动着至关重要的生命过程，如转录和调控。更广泛的影响：在支架晶体中精确控制功能分子的三维位置，为具有前所未有性能的材料打开了大门，可用于各种其他应用，包括生物传感、催化、能量转换、生物医学和生物技术。为了部分探索这些替代应用，该团队将为本科生领导的生物分子设计团队（2020年、2021年和2022年）提供3年的指导和资金，每个项目都将在国际BIOMOD竞赛中达到高潮。激励和培训下一代学生在生物材料设计前沿进行创新，将直接加快发现的步伐，造福科学界和国家。技术描述该团队将使用原子建模和模拟来设计“可扩展”蛋白质：DNA共晶结构。这种方法是实用的：重新设计已知形成共晶的蛋白质- dna系统。插入的DNA杆将被调整以保持现有的晶体接触和对称性。共晶将生长，优化，并稳定使用交联和连接化学。至关重要的是，在所得到的共晶中的大溶剂通道将允许结晶后的添加剂分子组装，使用插入的具有适当序列的DNA支柱进行同源DNA结合分子的位点特异性捕获。这种方法规避了传统晶体学的随机成核和生长。该团队将通过x射线衍射确定支架晶体捕获的dna结合分子是否可见。目标1。设计候选工程共晶。使用全原子模拟来优先考虑实验验证的变体。目标2。表达、纯化和结晶由工程蛋白和DNA构建块组成的新设计晶体。通过改变DNA块的长度和序列来优化晶体生长。目标3。使用化学交联稳定设计的共晶。捕获一个客体蛋白，它与插入到支柱中的DNA序列特异性结合。所提出的材料具有独特的方面，值得作为潜在的平台技术进行研究。[1]高多孔蛋白：DNA共晶体作为支架的设计和实验验证尚不清楚，[2]随后在其中捕获DNA结合蛋白的位点特异性也是未知的。与传统的生物分子晶体不同，所提出的支架晶体[3]的孔径可以通过改变DNA支柱中存在的碱基对的数量来扩大，[4]的拓扑结构可以适应任意DNA序列的模块化结合。该项目将侧重于这些材料的结构生物学应用，但侧面应用（如生物传感或催化）可能是3个本科生BIOMOD生物分子设计团队的重点，该项目将为他们提供指导和资金。该奖项反映了美国国家科学基金会的法定使命，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 DescriptionTo understand biology and to design medical interventions, it is essential to observe the structure adopted by large biological molecules. One of the main methods for determining those structures is to coax the molecules into arranging themselves into a crystal, and then to collect the diffraction pattern that results when these crystals are exposed to an X-ray beam. This conventional process has a high failure rate. Researchers have therefore long sought to develop scaffold crystals that can organize guest molecules.This project will produce a new class of candidate scaffold crystals. Technically, these new crystalline superstructures are co-crystals, since they are composed of both protein and DNA building blocks. To identify the most promising materials in this family, the team will use computational design and all-atom simulations to evaluate candidates for experimental validation. These new crystal materials will feature a modular design where “DNA struts” with any desired sequence are exposed to open channels that are large enough to permit the movement of guest proteins. The research team will grow crystals in which these DNA struts have sequences that are specifically recognized by certain DNA-binding proteins. When these guest proteins latch onto their target DNA they will join the crystal lattice and become observable via X-ray diffraction. This approach circumvents the haphazard crystal growth process that is the basis for conventional crystallography. The resulting materials may therefore provide a transformative method for scientists to routinely and easily observe atomic details for the complexes between DNA and DNA-binding proteins, complexes that drive critically important life processes such as transcription and regulation.Broader Impacts: Precise control of the 3-D position of functional molecules within a scaffold crystal opens the door for materials with unprecedented performance for diverse additional applications including biosensing, catalysis, energy conversion, biomedicine, and biotechnology. To partially explore these alternative applications, the team will provide mentorship and funding for 3 years of undergraduate-led biomolecular design teams (2020, 2021, and 2022), with each project culminating in the international BIOMOD competition. Inspiring and training the next generation of students to innovate at the biomaterials design frontier will directly accelerate the pace of discovery, to the benefit of the scientific community and the nation.Technical DescriptionThe team will use atomistic modeling and simulation to design “expandable” protein:DNA co-crystal structures. The approach is pragmatic: re-engineering protein-DNA systems that are already known to form co-crystals. Inserted DNA struts will be tuned to preserve existing crystallographic contacts and symmetry. Co-crystals will be grown, optimized, and stabilized using crosslinking and ligation chemistry. Critically, large solvent channels in the resulting co-crystals will permit post-crystallization additive molecular assembly, using the inserted DNA struts with the appropriate sequence for site-specific capture of cognate DNA-binding molecules. This approach circumvents the haphazard nucleation and growth that underlies conventional crystallography. The team will determine if DNA-binding molecules captured by the scaffold crystal become visible via X-ray diffraction. Goal 1. Design candidate engineered co-crystals. Use all-atom simulations to prioritize variants for experimental validation.Goal 2. Express, purify, and crystallize novel designed crystals composed of engineered protein and DNA building blocks. Optimize crystal growth by varying the length and sequence of the DNA blocks.Goal 3. Stabilize designed co-crystals using chemical crosslinking. Capture a guest protein that binds specifically to the DNA sequences that were inserted into the struts.The proposed materials have unique aspects that warrant investigation as a potential platform technology. [1] The design and experimental validation of highly porous protein:DNA co-crystals as scaffolds is unknown, as is [2] the subsequent site-specific capture of DNA-binding proteins therein. Unlike conventional biomolecular crystals, the proposed scaffold crystals [3] have pore sizes that may be expanded by changing the number of base pairs present within DNA struts, and [4] have topologies that are amenable to modular incorporation of arbitrary DNA sequences. This project will focus on the structural biology application of these materials, but side applications (e.g. biosensing or catalysis) may be the focus of the 3 annual undergraduate BIOMOD biomolecular design teams for whom the project will provide mentorship and funding.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.