Design of metal-mediated protein assemblies via hydroxamic acid functionalities

Rohit H. Subramanian, Jie Zhu, Jake B. Bailey, Jerika A. Chiong, Yiying Li, Eyal Golub, F. Akif Tezcan

Research output: Contribution to journalReview articlepeer-review

13 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)3264-3297
Number of pages34
JournalNature Protocols
Volume16
Issue number7
DOIs
StatePublished - Jul 2021
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature Limited.

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