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Design of metal-mediated protein assemblies via hydroxamic acid functionalities

Nature Protocols volume 16, pages 3264–3297 (2021)Cite this article

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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.

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Fig. 1: HA-mediated protein self-assembly.
Fig. 2: Design strategies for de novo protein self-assembly.
Fig. 3: Experimental overview for the generation of HA-mediated protein cages (Procedure 1).
Fig. 4: Experimental overview for the generation of HA-mediated protein-MOFs (Procedure 2).
Fig. 5: Selection of the protein building blocks for HA-mediated self-assembly.
Fig. 6: Synthetic schemes for the generation of HA ligands.
Fig. 7: Experimental setup and representative images of products in the synthesis of IHA.
Fig. 8: Anticipated results for HA-mediated protein self-assembly.

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Data availability

The principal data supporting the findings of this work are available within the figures and the Supplementary Information. Additional data that support the findings of this study are available from the corresponding author on request.

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Acknowledgements

We thank N. Avakyan and R. Alberstein for helpful discussions. This work was supported by the US Department of Energy (Division of Materials Sciences, Office of Basic Energy Sciences; DE-SC0003844; for protein-MOF work, including design, data collection and analysis as well as for protein-cage design and EM analyses) and by the National Science Foundation (Division of Materials Research; DMR-1602537 and DMR-2004558; crystallographic analyses of protein cages). R.H.S. was supported by the National Institute of Health Chemical Biology Interfaces Training Grant UC San Diego (T32GM112584).

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA, USA

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

  2. Materials Science and Engineering, University of California, San Diego, La Jolla, CA, USA

    F. Akif Tezcan

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Contributions

E.G. and F.A.T. conceived the protein-cage project. J.Z., J.A.C. and Y.L. synthesized HA ligands for the protein-cage project. E.G. and R.H.S. performed protein-cage experiments and data analysis. J.B.B. and F.A.T. conceived the protein-MOF project. J.B.B. synthesized ditopic HA ligands and performed protein-MOF experiments and data analysis. R.H.S. and F.A.T. wrote the manuscript with contributions from all authors.

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Correspondence to F. Akif Tezcan.

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Key references using this protocol

Sontz, P. A. et al. J. Am. Chem. Soc. 137, 11598–11601 (2015): https://pubs.acs.org/doi/10.1021/jacs.5b07463

Bailey, J. B. et al. J. Am. Chem. Soc. 139, 8160–8166 (2017): https://pubs.acs.org/doi/10.1021/jacs.7b01202

Golub, E. et al. Nature 578, 172–176 (2020): https://www.nature.com/articles/s41586-019-1928-2

Bailey, J. B. et al. J. Am. Chem. Soc. 142, 17265–17270 (2020): https://pubs.acs.org/doi/10.1021/jacs.0c07835

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Subramanian, R.H., Zhu, J., Bailey, J.B. et al. Design of metal-mediated protein assemblies via hydroxamic acid functionalities. Nat Protoc 16, 3264–3297 (2021). https://doi.org/10.1038/s41596-021-00535-z

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