The self-assembly of proteins into sophisticated multicomponent assemblies is a hallmark of all living systems and has spawned extensive efforts in the construction of novel synthetic protein architectures with emergent functional properties. Protein assemblies in nature are formed via selective association of multiple protein surfaces through intricate noncovalent protein–protein interactions, a challenging task to accurately replicate in the de novo design of multiprotein systems. In this protocol, we describe the application of metal-coordinating hydroxamate (HA) motifs to direct the metal-mediated assembly of polyhedral protein architectures and 3D crystalline protein–metal–organic frameworks (protein-MOFs). This strategy has been implemented using an asymmetric cytochrome cb₅₆₂ monomer through selective, concurrent association of Fe³⁺ and Zn²⁺ ions to form polyhedral cages. Furthermore, the use of ditopic HA linkers as bridging ligands with metal-binding protein nodes has allowed the construction of crystalline 3D protein-MOF lattices. The protocol is divided into two major sections: (1) the development of a Cys-reactive HA molecule for protein derivatization and self-assembly of protein–HA conjugates into polyhedral cages and (2) the synthesis of ditopic HA bridging ligands for the construction of ferritin-based protein-MOFs using symmetric metal-binding protein nodes. Protein cages are analyzed using analytical ultracentrifugation, transmission electron microscopy and single-crystal X-ray diffraction techniques. HA-mediated protein-MOFs are formed in sitting-drop vapor diffusion crystallization trays and are probed via single-crystal X-ray diffraction and multi-crystal small-angle X-ray scattering measurements. Ligand synthesis, construction of HA-mediated assemblies, and post-assembly analysis as described in this protocol can be performed by a graduate-level researcher within 6 weeks.

蛋白质自组装成复杂的多组分组件是所有生命系统的标志，并且已经在构建具有新兴功能特性的新型合成蛋白质结构方面产生了广泛的努力。自然界中的蛋白质组装体是通过复杂的非共价蛋白质-蛋白质相互作用，通过多个蛋白质表面的选择性结合形成的，这是在多蛋白质系统的从头设计中准确复制的一项具有挑战性的任务。在本协议中，我们描述了金属配位异羟肟酸盐 (HA) 基序在指导多面体蛋白质结构和 3D 结晶蛋白质-金属-有机框架（蛋白质-MOF）的金属介导组装中的应用。该策略已使用不对称细胞色素cb _562实施^{通过 Fe 3+}和 Zn ²⁺的选择性并发结合形成的单体离子形成多面体笼。此外，使用双位 HA 接头作为桥接配体与金属结合蛋白节点已经允许构建结晶 3D 蛋白-MOF 晶格。该方案分为两个主要部分：(1) 开发用于蛋白质衍生化和蛋白质-HA 结合物自组装成多面体笼的 Cys 反应性 HA 分子，以及 (2) 用于构建的双位 HA 桥接配体的合成使用对称金属结合蛋白节点的基于铁蛋白的蛋白质-MOFs。使用分析超速离心、透射电子显微镜和单晶 X 射线衍射技术分析蛋白质笼。HA 介导的蛋白质-MOF 在坐滴蒸汽扩散结晶托盘中形成，并通过单晶 X 射线衍射和多晶小角 X 射线散射测量进行探测。研究生级别的研究人员可以在 6 周内完成本协议中描述的配体合成、HA 介导的程序集的构建和组装后分析。