Designed protein self-assembly by autocatalytic isopeptide bond formation

通过自催化异肽键形成设计蛋白质自组装

• 批准号: 502051463
• 负责人: Dr. Ulrich Markel
• 金额: --
• 依托单位: Department of Chemistry and Biochemistry
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2022
• 资助国家: 德国
• 起止时间: 2021-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/502051463?language=en
• 关键词: Designed; protein; self; assembly; autocatalytic

Owing to their structural diversity, proteins perform a variety of functions in living systems from catalysis to serving as nature’s building block for the assembly of dynamic materials and molecular machines. Inspired by these natural examples, researchers have sought ways to design protein assemblies from discrete oligomeric structures to higher-order structures (e.g., 1D fibers, 2D arrays, and 3D lattices) following different non-covalent and covalent assembly strategies. Isopeptide bonds (IPB) are a particularly intriguing example of how nature utilizes covalent bonds to build higher-order protein structures and equip them with exceptional stability. Yet, these natural IPB-forming systems are limited to few protein classes. Previously, engineered variants of these natural IPB-forming systems have been used as genetic fusion tags to drive autocatalytic protein assembly. But these systems are derived from pathogenic bacteria and always leave a molecular scar, which can prompt unwanted immune responses in potential therapeutic applications. Being able to rationally design autocatalytic IPB into a protein-protein interface of choice would thus be highly desirable. This project aims to i) rationalize de novo IPB design in proteins that are structurally unrelated to natural IPB-forming systems and ii) utilize the de novo design of IPB for the assembly of discrete and higher-order protein assemblies. To this end, the homodimeric four-helix bundle protein Rop will be used as proof-of-principle. The four-helix architecture represents a common motif that is often observed independently and as a subunit of larger protein folds and is structurally unrelated to any natural IPB-forming system. A combined approach of computational design and directed evolution will be used to establish homo- and heterodimeric protein assemblies with different numbers of IPB, thereby providing tunable levels of assembly stabilization. Using these discrete assemblies as building blocks, IPB formation will be used to build higher-order structures with high levels of covalent interconnectivity as well as 1D assemblies (i.e., linear protein polymers), both of which are expected to possess exceptional structural stability. Finally, the IPB design principles established in the initial phase of the project will be transferred to other proteins to test the generality of the approach. This could lead to interesting new applications. For instance, by combining the structure-stabilizing features of IPB with orthogonal and switchable protein assembly strategies (e.g., metal-templated assembly or disulfide bonds), stable but stimuli responsive nanostructures with potential therapeutic relevance are envisioned. Overall, the proposed project will demonstrate the de novo design of IPB as a novel means to establish structure-stabilizing covalent interactions that will drive the next generation of protein assemblies.

由于它们的结构多样性，蛋白质在从催化到充当动态材料和分子机器组装的自然基础的生活系统中发挥了多种功能。受这些自然示例的启发，研究人员寻求方法来设计蛋白质组件，从离散的寡聚结构到高阶结构（例如1D纤维，2D阵列和3D晶格），遵循不同的非共价和共价组装策略。异肽键（IPB）是一个特别有趣的例子，说明了自然如何利用共价键来建立高阶蛋白质结构，并为它们配备出色的稳定性。然而，这些天然IPB形成系统仅限于少数蛋白质类别。以前，这些天然IPB形成系统的工程变体已被用作遗传融合标签，以驱动自催化蛋白质组件。但是这些系统源自致病细菌，并始终留下分子疤痕，这可能会在潜在的治疗应用中促使不需要的免疫复杂。因此，能够将自催化的IPB合理设计为选择的蛋白质 - 蛋白质界面是非常可取的。该项目旨在i）在结构上与天然IPB形成系统无关的蛋白质中，从头开始IPB设计，ii）利用IPB的从头设计来组装离散和高阶蛋白质组件。为此，同二聚体四螺旋束蛋白ROP将用作原理证明。四螺旋结构代表了一个常见的基序，通常是独立观察的，是较大蛋白质折叠的亚基，并且在结构上与任何天然IPB形成系统无关。计算设计和定向进化的组合方法将用于建立具有不同数量IPB的同型和异二聚体蛋白质组件，从而提供可调的装配稳定水平。使用这些离散的组件作为构建块，IPB的形成将用于建立具有高水平的共价互连性以及1D组件（即线性蛋白聚合物）的高阶结构，这两种结构都预计可能具有潜在的特殊结构稳定性。最后，在项目初始阶段建立的IPB设计原理将转移到其他蛋白质中，以测试方法的通用性。这可能会导致有趣的新应用程序。例如，通过将IPB的结构稳定特征与正交和可切换蛋白质组装策略（例如金属构成的组件或二硫键键）相结合，可以设想稳定但刺激的纳米结构与潜在的治疗性相关性。总体而言，拟议的项目将证明IPB的从头设计是一种新型手段，以建立结构稳定的共价相互作用，这将推动下一代蛋白质组件。