RAISE: Design of co-assembling peptides as recombinant protein fusion tags for integrating enzymes into supramolecular hydrogels

该RAISE项目由工程理事会CBET部门的纳米材料生物与环境相互作用项目、生物科学理事会分子与细胞生物科学部的分子生物物理学项目和综合活动办公室共同资助。在这个多学科的项目中，纳米材料支架，或水凝胶，固定化酶将使用蛋白质作为设计模板。该方法为传统的固定技术提供了一种新的选择，传统的固定技术更困难和耗时。这种新颖的组装技术被设计成可以选择性地自组装成纳米纤维结构。将蛋白质定向设计和组装成纳米纤维是一个令人兴奋的新研究前沿，在基本蛋白质组装和功能纳米材料的设计方面，这一研究前沿在很大程度上是未被探索的。一个多学科的研究团队将结合他们在建模、成像和表征方法方面的专业知识，以展示新的蛋白质组装，并推进对蛋白质组装的基本理解。所提出的技术将是一个非凡的改进，目前最先进的蛋白质组装方法。该技术的成功实施预计将对各种生物医学和生物技术应用的新型功能材料的工程产生长期广泛的影响。各种各样的教育活动将被开发，包括一个关于蛋白质折叠的iPad应用程序，以及一个描述如何使用计算方法来设计新的生物材料的视频。所提出的方法绕过了传统的固定化技术，其中纳米结构支架通过其表面的反应在组装后被功能化。相反，酶是由细菌通过带有肽融合标签的重组DNA来表达的，这些标签介导它们组装成纳米结构的材料。该方法的关键是使用融合标签，当与互补肽混合时，融合标签可共同组装成纤维结构，但在纯时仍未组装。在蛋白质生物物理学和功能纳米材料领域，共组装肽是一个令人兴奋的研究前沿。该项目的目标是展示新型酶功能化的超分子水凝胶，并推进预测肽共组装所需的基本生物物理理解。提出了三个具体目标：(1)表征已知的<s:1>纳米片纤维形成共组装肽对的组装过程和最终结构；(2)设计和测试新的选择性共组装肽对；(3)测试共组装肽作为纳米结构生物材料中固定蛋白质的标签。每个目标都将利用PI Hall的计算建模专业知识，Co-PI Hudalla提供的肽纳米纤维的光谱和微观表征专业知识，以及Co-PI Paravastu提供的固态核磁共振专业知识。所提出的酶固定方法将是对目前最先进的蛋白质固定方法的非凡改进，因此为解决将生物活性折叠蛋白质安装到生物材料中的长期挑战打开了大门。各种各样的教育活动将被开发，包括一个关于蛋白质折叠的iPad应用程序，以及一个描述如何使用计算方法来设计新的生物材料的视频。

This RAISE project is jointly funded by the Biological and Environmental Interactions of Nanoscale Materials program in CBET Division in the Engineering Directorate, the Molecular Biophysics Program in the Division of Molecular and Cellular Biosciences in the Biological Sciences Directorate, and the Office of Integrative Activities. In this multi-disciplinary project, nanomaterial scaffolds, or hydrogels, with immobilized enzymes will be produced using proteins as a design template. The proposed method provides a novel alternative to conventional immobilization techniques which are more difficult and time consuming. This novel assembly technique is designed so that it selectively self-assembles into nano fiber structures. This targeted design and assembly of proteins into nanofibers is an exciting new research frontier that is largely unexplored with regard to both fundamental protein assembly and design of functional nanomaterials. A multi-disciplinary team of investigators will combine their expertise in modeling, imaging and characterization methods in order to demonstrate novel protein assembly and to advance fundamental understanding of protein assembly. The proposed technique will be an extraordinary improvement over the current state-of-the-art in protein assembly methods. Successful implementation of the proposed technique is anticipated to have a long-term broad impact on the engineering of novel functional materials for various biomedical and biotechnological applications. A variety of educational activities will be developed, including an iPad app on protein folding, and a video describing how computational methods can be used to design new biomaterials.The proposed method bypasses conventional immobilization techniques in which the nanostructured scaffold is functionalized post-assembly via reactions at its surface. Instead, enzymes are expressed by bacteria from recombinant DNA with peptide fusion tags that mediate their assembly into nanostructured materials. The key to the method is the use of fusion tags that co-assemble into fibrillar structures when mixed with a complementary peptide, but remain unassembled when pure. Co-assembling peptides are an exciting research frontier that is virtually unexplored in both protein biophysics and functional nanomaterials. The goals of the project are to demonstrate novel enzyme-functionalized supramolecular hydrogels, and to advance fundamental biophysical understanding necessary to predict peptide co-assembly. Three specific aims are proposed: (1) characterize the assembly process and resultant structure for known â-sheet nanofiber-forming co-assembling peptide pairs, (2) design and test new pairs of selectively co-assembling peptides and, (3) test co-assembling peptides as tags to immobilize proteins in nanostructured biomaterials. Each aim will leverage the computational modeling expertise of PI Hall, with expertise in spectroscopic and microscopic characterization of peptide nanofibers provided by Co-PI Hudalla and solid-state nuclear magnetic resonance expertise provide by Co-PI Paravastu. The proposed enzyme immobilization method will be an extraordinary improvement over the current state-of-the-art in protein immobilization methods and hence opens the door to addressing long-standing challenges in installing biologically-active, folded proteins into biomaterials. A variety of educational activities will be developed, including an iPad app on protein folding, and a video describing how computational methods can be used to design new biomaterials.