Controlling protein assembly on inorganic crystals through designed protein interfaces

Abstract

The ability of proteins and other macromolecules to interact with inorganic surfaces is essential to biological function. The proteins involved in these interactions are highly charged and often rich in carboxylic acid side chains1,2,3,4,5, but the structures of most protein–inorganic interfaces are unknown. We explored the possibility of systematically designing structured protein–mineral interfaces, guided by the example of ice-binding proteins, which present arrays of threonine residues (matched to the ice lattice) that order clathrate waters into an ice-like structure6. Here we design proteins displaying arrays of up to 54 carboxylate residues geometrically matched to the potassium ion (K+) sublattice on muscovite mica (001). At low K+ concentration, individual molecules bind independently to mica in the designed orientations, whereas at high K+ concentration, the designs form two-dimensional liquid-crystal phases, which accentuate the inherent structural bias in the muscovite lattice to produce protein arrays ordered over tens of millimetres. Incorporation of designed protein–protein interactions preserving the match between the proteins and the K+ lattice led to extended self-assembled structures on mica: designed end-to-end interactions produced micrometre-long single-protein-diameter wires and a designed trimeric interface yielded extensive honeycomb arrays. The nearest-neighbour distances in these hexagonal arrays could be set digitally between 7.5 and 15.9 nanometres with 2.1-nanometre selectivity by changing the number of repeat units in the monomer. These results demonstrate that protein–inorganic lattice interactions can be systematically programmed and set the stage for designing protein–inorganic hybrid materials.

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Fig. 1: Design and characterization of lattice-matched protein monomers.
Fig. 2: Assembly of DHR10-micaX molecules on mica (001).
Fig. 3: Liquid crystals formed by single-protein-diameter DHR-micaX-NC nanowires.
Fig. 4: Protein–protein interactions drive formation of the tunable hexagonal lattice.

Data availability

Design models in PDB format are available on Github (https://github.com/pylesharley/DHR10micaX/tree/master/rosetta_models). Source data for Supplementary Figs. 26 are provided with the paper. All other data not included in the manuscript are available upon reasonable request from the corresponding authors.

Code availability

The Rosetta Macromolecular Modelling suite is available for non-commercial use at (https://www.rosettacommons.org). The specific Rosetta applications used were Rosetta Scripts46, Remodel47, and Pyrosetta48. Foldit33, a graphic user interface to Rosetta, was used as well. PyMOL49 was used to view the design models and prepare input files for Rosetta protocols.

A Github repository (https://github.com/pylesharley/DHR10micaX) contains the Rosetta protocols, input files and Python scripts used to model the protein assembles on mica, and corresponding README.txt files with instructions.

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Acknowledgements

We thank T. Brunette, P. Huang, F. Parmeggiani, Y. Hsia, W. Sheffler, T. Craven, S. Boyken and Z. Chen for suggestions; D. Alonso, L. Goldschmidt and P. Vecchiato for supporting computational resources; B. Legg and J. Tao for AFM support; B. Legg for providing the model of mica (001); L. Carter for size-exclusion chromatography with multi-angle light scattering (SEC-MALS) support. F. Busch and V. H. Wysocki provided native mass spectrometry support (NIH National Institute of General Medical Sciences award number P41GM128577). Development of the imaging protocols was supported by the Laboratory Directed Research and Development Office through the Materials Synthesis and Simulations Across Scales Initiative at Pacific Northwest National Laboratory (PNNL). AFM experiments on the DHR10-micaX and DHR10-micaX-NC were supported by the US Department of Energy (DOE), the Office of Basic Energy Sciences (BES), Biomolecular Materials Program (BMP) at PNNL. AFM experiments on DHR10-micaX-H formation of hexagonal lattices and analysis of DHR10-micaX binding kinetics were performed at PNNL and supported by the US DOE BES Energy Frontier Research Center CSSAS (The Center for the Science of Synthesis Across Scales) located at the University of Washington (award number DE-SC0019288). Design and synthesis of DHR10-micaX-H and its variants were performed at the University of Washington and supported by the US DOE BES BMP (award number DE-SC0018940). H.P. was supported by the Institute for Protein Design Materials Science Research Gift Fund and Michelson Medical Research Foundation, Protein Design Initiative Fund, and DOE Biomolecular Materials Program (award number DE-SC0018940). D.B. is funded by the Howard Hughes Medical Institute and Bruce and Jeannie Nordstrom / Patty and Jimmy Barrier Gift for the Institute for Protein Design Directors Fund. We thank the staff at the Advanced Light Source SIBYLS beamline at Lawrence Berkeley National Laboratory, including K. Burnett, G. Hura and J. Tainer for the services provided through the mail-in SAXS programme, which is supported by the DOE Office of Biological and Environmental Research Integrated Diffraction Analysis program DOE BER IDAT grant (award number DE-AC02-05CH11231) and NIGMS supported ALS-ENABLE (award number GM124169-01). PNNL is a multi-programme national laboratory operated for the Department of Energy by Battelle under contract number DE-AC05-76RL01830.

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Nature thanks Roberto A. Chica, Rajesh R. Naik and Sarah S. Staniland for their contribution to the peer review of this work.

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Contributions

H.P. designed, expressed, and purified the proteins. S.Z. developed the protocol to assemble protein on mica and performed the AFM experiments. S.Z. and J.J.D.Y. analysed the AFM data. D.B. and J.J.D.Y. supervised the project. All authors wrote the manuscript.

Corresponding authors

Correspondence to James J. De Yoreo or David Baker.

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Supplementary Information

Supplementary Figures 1-29 and Supplementary Tables 1-2 are in the Supplementary Information pdf, arranged so that each Supplementary Figure is on the page corresponding to its number. Table S1 is on page 10 with related Figure S10. Table S2 contains amino-acid sequences and is on pages 30-33.

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Pyles, H., Zhang, S., De Yoreo, J.J. et al. Controlling protein assembly on inorganic crystals through designed protein interfaces. Nature 571, 251–256 (2019). https://doi.org/10.1038/s41586-019-1361-6

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