Shaping the Future of Protein Engineering - Biochemistry (ACS

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Shaping the Future of Protein Engineering Dominic J. Glover,† Dawei Xu,‡ and Douglas S. Clark*,‡ †

School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, NSW 2052, Australia Department of Chemical and Biomolecular Engineering, University of California, Berkeley, California 94720, United States



the filaments. This was achieved by varying the number of helical repeat units in the monomer building blocks, with the diameter of the filament changing in a linear manner with the number of repeat units. The assembly of the filaments could also be modulated by the creation of capping proteins that enable control over filament length. These capping proteins lack one of the two designed protein−protein interfaces, which prevents further elongation upon incorporation into the filament termini. Incubating the capping proteins with already assembled filaments resulted in a decrease in filament length, suggesting the subunits in the filaments are dynamically exchanged over time. Ultimately, the capping proteins may serve additional useful proposes such as enabling the attachment of functional molecules to the ends of filaments or joining individual capped filaments together. A similar strategy of using filament-capping proteins has been used previously to connect filaments into branched assemblies.5 The material properties of protein fibers and related constructs remain difficult to predict with a high degree of certainty, regardless of how they may be engineered and assembled. However, this too will become more feasible as the art and science of protein engineering continue to evolve and as physicochemical techniques for characterizing proteins continue to advance. Likewise, the computational design of two-dimensional protein structures that self-assemble and then orient properly and controllably assemble into more elaborate three-dimensional structures cannot be very far off. Such systems will create many exciting opportunities as new biomaterials and biomechanical devices by serving as structural architectures upon which to position, join, or encapsulate functional materials (Figure 2). For example, modifiable filamentous assemblies have been used as templates to align metal nanoclusters to create electrically conductive nanowires for possible nanoelectronics applications.5 Alternatively, protein cage nanostructures with sufficiently large internal openings can be used for the compartmentalization of enzymes for control and enhancement of catalytic activity. If the central cavity of tubular assemblies such as those created by Shen et al. could be suitably modified, then elegant enzyme cascades could be envisaged whereby individual enzymes are enclosed in a tube that prevents metabolic intermediates from diffusing away before reacting with sequential enzymes. Designing the protein interfaces of these nanostructures to enable controlled assembly and disassembly, for example by pH or redox state, will enable vehicles to be created for controlled delivery and release of cargo or for sensing and signal transduction. There is also the potential to exploit or mimic the mechanical activity of

n the early days of “protein engineering”, the late Jeremy Knowles wrote an article entitled Tinkering with Enzymes: What Are We Learning?,1 in which he remarked “I have argued in vain against the premature use of the word engineering, on the grounds that an engineer knows what he is doing when he designs a bridge, whereas the protein chemist has yet to define the equivalents of Hooke’s law, Young’s modulus, and the rest.” Although much progress has been made since the time of this incisive quote toward harnessing the potential of proteins as building blocks for the construction of tailor-made scaffolds and templates with specific functions,2,3 Knowles’ point about the apparent misnomer of protein engineering has remained largely justified. However, thanks to the recent groundbreaking work of Shen et al.,4 we now have an example of engineering proteins that comes much closer to meeting Knowles’ criteria. While it may still not be possible to design and build the bridge, it is now at least possible to design and construct the cables. Earlier advances in the design of protein nanostructures focused on redesigning already existing interfaces within symmetrical protein building blocks. In contrast, natural protein complexes generally consist of independently folded and asymmetric proteins, which endows a level of dynamic assembly or disassembly in response to changing physiological conditions. In this new design paradigm of Shen et al., a computational approach was developed to design multiple protein interfaces in monomeric building blocks for assembly into dynamic helical filaments on the micrometer scale (Figure 1). The approach begins with an arbitrary monomer protein structure consisting of repeating helical domains. A second copy of the monomer is generated in a random orientation and moved toward the first copy until they come into contact. After the creation of this interfacial contact, the computational design proceeds by sampling multiple ways to build symmetrical helix geometries. During this process, various assembly parameters are examined, with the goal of generating several repeating turns of the full filament. Successful filament designs with evenly distributed interfaces are selected, and combinatorial sequence optimization of the protein−protein interfaces is performed on a central monomer in the filament, propagating the amino acid sequence to all other monomers. Moving from a computer to the lab bench, the authors produced the proteins recombinantly, with subsequent imaging by electron microscopy revealing that many of the designs produced filamentous assemblies in high yields. The fabrication of scaffolds and templates for nanomaterials will require precise control over both the dimensions and the assembly process of the underlying protein building blocks. Setting their work apart from previous filament engineering was the ease with which Shen et al. could vary the diameter of

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© XXXX American Chemical Society

Received: December 31, 2018

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DOI: 10.1021/acs.biochem.8b01322 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Computational design process and cryo-electron microscopy-resolved filament architecture. Adapted from ref 4. Cryo-EM accession code EMD-9017 and Protein Data Bank (PDB) entry 6E9T for the filament.

Figure 2. Hypothetical application of protein nanofibers as electronically conductive wires.



protein complexes such as F1-ATPase to create biological nanomachines ranging from levers and rotors to motor-driven assemblies. As the recent work of Shen et al. demonstrates, the field of protein engineering is no longer in a state of empirical infancy. It has advanced from the tinkering stage described by Knowles to a level of sophistication whereby protein−protein interactions can be designed from scratch, leading to the assembly of higher-order structures of predictable size and conformation. As time passes and additional design hurdles are overcome, the field will continue to assume more of the qualities that define other fields of engineering, in which codified principles are used to guide the design, construction, and operation of structures, devices, and processes. Protein engineering will have come of age, in the true sense of the word.

AUTHOR INFORMATION

Corresponding Author

*Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720. E-mail: dsc@ berkeley.edu. Phone: 510-642-2408. Fax: 510-643-1228. ORCID

Douglas S. Clark: 0000-0003-1516-035X Funding

This work was supported by the Air Force Office of Scientific Research (FA9550-17-1-0451). Notes

The authors declare no competing financial interest. B

DOI: 10.1021/acs.biochem.8b01322 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry



REFERENCES

(1) Knowles, J. R. (1987) Tinkering with enzymes: what are we learning? Science 236, 1252−1258. (2) Lai, Y.-T., Reading, E., Hura, G. L., Tsai, K.-L., Laganowsky, A., Asturias, F. J., Tainer, J. A., Robinson, C. V., and Yeates, T. O. (2014) Structure of a designed protein cage that self-assembles into a highly porous cube. Nat. Chem. 6, 1065−1071. (3) Bale, J. B., Gonen, S., Liu, Y., Sheffler, W., Ellis, D., Thomas, C., Cascio, D., Yeates, T. O., Gonen, T., King, N. P., and Baker, D. (2016) Accurate design of megadalton-scale two-component icosahedral protein complexes. Science 353, 389−394. (4) Shen, H., Fallas, J. A., Lynch, E., Sheffler, W., Parry, B., Jannetty, N., Decarreau, J., Wagenbach, M., Vicente, J. J., Chen, J., Wang, L., Dowling, Q., Oberdorfer, G., Stewart, L., Wordeman, L., De Yoreo, J., Jacobs-Wagner, C., Kollman, J., and Baker, D. (2018) De novo design of self-assembling helical protein filaments. Science 362, 705−709. (5) Glover, D. J., Giger, L., Kim, S. S., Naik, R. R., and Clark, D. S. (2016) Geometrical assembly of ultrastable protein templates for nanomaterials. Nat. Commun. 7, 11771.

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DOI: 10.1021/acs.biochem.8b01322 Biochemistry XXXX, XXX, XXX−XXX