Design of metal-mediated protein assemblies via hydroxamic acid functionalities
Design of metal-mediated protein assemblies via hydroxamic acid functionalities
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DOI：
10.1038/s41596-021-00535-z
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发表时间：
2021-05-28
期刊：
影响因子：
14.8
通讯作者：
Tezcan，F. Akif
中科院分区：
文献类型：
--
作者：
Subramanian，Rohit H.;Zhu，Jie;Tezcan，F. Akif
蛋白质自组装成复杂的多组分组装体是所有生命系统的标志，并在构建具有紧急功能特性的新型合成蛋白质架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缔合形成的，这是一项具有挑战性的任务，需要在多蛋白质系统的从头设计中准确复制。在该协议中，我们描述了金属配位异羟肟酸（HA）基序的应用，以指导金属介导的多面体蛋白质结构和3D晶体蛋白质-金属-有机框架（蛋白质-MOFs）的组装。这种策略已经实施使用不对称的细胞色素b562单体，通过选择性，并发协会的Fe 3+和Zn 2+离子，形成多面体笼。此外，使用双位HA接头作为具有金属结合蛋白节点的桥接配体已经允许构建结晶3D蛋白质-M0 F晶格。该方案分为两个主要部分：（1）开发Cys反应性HA分子，用于蛋白质衍生化和蛋白质-HA缀合物自组装成多面体笼，以及（2）合成双位HA桥接配体，用于使用对称金属结合蛋白质节点构建基于铁蛋白的蛋白质-M0 F。使用分析超离心、透射电子显微镜和单晶X-射线衍射技术分析蛋白笼。HA介导的蛋白质-MOFs在坐滴气相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。配体合成，HA介导的组装体的构建，以及本方案中描述的组装后分析可以由研究生水平的研究人员在6周内进行。
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 cytochromecb562monomer 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.