DMREF: Collaborative Research: Helical Protein Assemblies by Design

非技术总结分子自组装是生命的基本原则，细胞已经掌握了这一过程，以编码令人难以置信的多样性功能。螺旋蛋白质组装体组织大部分细胞内和细胞外结构，并指导所有运动。通过设计合成蛋白质组装体来模拟这些功能的能力将改变现代分子科学，具有深远的应用，包括运动，控释，定向运输，动态切换和形状选择性催化。然而，在纳米尺度上的结构有序的超分子材料是最具挑战性的合理构建和结构分析最困难的。这些扩展的蛋白质组装体的大小和结构复杂性对当前的计算设计方法提出了重大挑战。控制蛋白质-蛋白质相互作用的规则比DNA更复杂，更难以可靠地预测。在这个项目中，一个新的智能框架，有针对性地设计合成蛋白质组装在原子级的精度将被建立，验证，并提供给研究界。通过三位研究人员的综合专业知识，这种方法将把建模和计算设计方面的重大进展与前所未有的实验技术结合起来，用于在原子水平上确定蛋白质组装体的结构。在发展这一框架的道路上，将解决对生物学、化学和材料科学具有重大意义的基本问题：从发展对原生生物组装体功能作用的理解，到构建用于技术应用的合成组装体。学生（研究生和本科生），博士后和教师参与本项目将获得各种计算，合成和分析方法的基本技术兴趣的研究领域的经验，这将使他们为未来的科学事业做好准备。三个学术机构之间的交流项目（埃默里大学，弗吉尼亚大学，和达特茅斯学院）将成立，这将允许学生和博士后成为参与这个研究项目的不同方面。用更简单、更易处理的合成材料捕捉这些分子，将是分子科学的一次重大飞跃。基因组测序，生物信息学分析，近原子分辨率cryo-EM结构测定和计算蛋白质设计的最新技术进展，与现存的合成和分析方法相结合，提出了一个前所未有的机会，工程师新的蛋白质组装，模仿和改善其天然的对应部分。该项目将采用基于天然结构表示的蛋白质可设计性，作为促进和控制折叠蛋白质基序之间关联的机制，旨在创建具有特定结构和功能的蛋白质材料。蛋白质工程背景下的可设计性是指蛋白质折叠在序列空间中的稳健性。可设计性的代表是蛋白质数据库（PDB）中结构基序的出现频率。这种方法将被用来寻找蛋白质结构数据库中的原聚体之间的可设计界面。拟议研究的最终目标将是定义基于简单的二级或三级结构元素的序列，这些结构元素能够自组装成具有扩展螺旋对称性的纳米级材料。将采用计算方法来询问蛋白质结构数据库，以确定稳健的结构基序内的可设计界面。将计算优化和合成合适的候选序列。最初将采用生物物理学方法来鉴定具有有希望的自组装行为的序列。将使用来自冷冻EM图像的迭代螺旋实空间重建（IHRSR）对这些组件进行最先进的高分辨率结构分析。显着的改进，成像硬件，重建算法，和计算方法的结构细化提供了快速访问近原子分辨率的结构的本地和合成的螺旋组装。这些分析将为未来的计算建模和设计提供信息，从而在理论、综合和先进的结构分析方法之间建立一个动态反馈回路。

NON-TECHNICAL SUMMARYMolecular self-assembly is a fundamental principle of life, with cells having mastered this process to encode incredible diversity of function. Helical protein assemblies organize much of the intracellular and extracellular structure, and direct all movement. The ability to emulate such functions by designing synthetic protein assemblies would transform modern molecular science, with far-reaching applications including locomotion, controlled release, directional transport, dynamic switching, and shape-selective catalysis. However, structurally ordered supramolecular materials on the nanometer length-scale are the most challenging to rationally construct and the most difficult to structurally analyze. The size and structural complexity of these extended protein assemblies present a significant challenge to current computational design methods. The rules that govern protein-protein interactions are more complex and difficult to reliably predict than for DNA. In this project, a novel intellectual framework for the targeted design of synthetic protein assemblies at atomic-level accuracy will be established, validated, and made available to the research community. Enabled by the combined expertise of the three investigators involved, this approach will merge significant advances in modeling and computational design with never-before-possible experimental techniques for structural determination of protein assemblies at the atomic level. On the way to developing this framework, fundamental questions of acute significance to biology, chemistry, and materials science will be addressed: from development of an understanding of the functional roles of native biological assemblies to construction of synthetic assemblies for technological applications. Students (graduate and undergraduate), postdoctorals, and faculty involved in this project will gain experience in a variety of computational, synthetic, and analytical methods in research areas of fundamental technological interest that will prepare them well for future scientific careers. An exchange program between the three academic institutions (Emory University, University of Virginia, and Dartmouth College) will be established that will permit students and postdoctorals to become involved in the different aspects of this research project.TECHNICAL SUMMARYHelical protein assemblies in biological systems exhibit a rich portfolio of structure and function; capturing these within simpler and more tractable synthetic materials would amount to a major leap in molecular science. Recent technological advances in genome sequencing, bioinformatic analysis, near-atomic resolution cryo-EM structural determination, and computational protein design, in combination with extant synthetic and analytical methods, present an unprecedented opportunity to engineer novel protein assemblies that emulate and improve upon their native counter-parts. This project will employ protein designability, estimated on the basis of native structural representation, as a mechanism to promote and control association between folded protein motifs with an aim to create protein-based materials of defined structure and function. Designability in the context of protein engineering refers to robustness of a protein fold in sequence space. A proxy for designability is the frequency of occurrence of a structural motif within the Protein Data Bank (PDB). This approach will be employed to search for designable interfaces between protomers within the protein structural databank. The ultimate objective of the proposed research will be to define sequences based on simple secondary or tertiary structural elements that are competent for self-assembly into nano-scale materials with extended helical symmetry. Computational methods will be employed to interrogate the protein structural databank to identify designable interfaces within robust structural motifs. Suitable candidate sequences will be computationally optimized and synthesized. Proven biophysical methods will be employed initially to identify sequences with promising self-assembly behavior. State-of-the-art high-resolution structural analyses will be performed on these assemblies using Iterative Helical Real-Space Reconstruction (IHRSR) from cryo-EM images. Dramatic improvements in imaging hardware, reconstruction algorithms, and computational methods of structural refinement have provided rapid access to near-atomic resolution structures of native and synthetic helical assemblies. These analyses will inform future rounds of computational modeling and design, thus establishing a dynamic feedback loop between theory, synthesis, and advanced methods of structural analysis.