Issue 1, 2022

Heterogeneous protein co-assemblies with tunable functional domain stoichiometry

Abstract

In nature, the precise heterogeneous co-assembly of different protein domains gives rise to supramolecular machines that perform complex functions through the co-integrated activity of the individual protein subunits. A synthetic approach capable of mimicking this process would afford access to supramolecular machines with new or improved functional capabilities. Here we show that the distinct peptide strands of a heterotrimeric α-helical coiled-coil (i.e., peptides “A”, “B”, and “C”) can be used as fusion tags for heterogeneous co-assembly of proteins into supramolecular structures with tunable subunit stoichiometry. In particular, we demonstrate that recombinant fusion of A with NanoLuc luciferase (NL-A), B with superfolder green fluorescent protein (sfGFP-B), and C with mRuby (mRuby-C) enables formation of ternary complexes capable of simultaneously emitting blue, green, and red light via sequential bioluminescence and fluorescence resonance energy transfer (BRET/FRET). Fusion of galectin-3 onto the C-terminus of NL-A, sfGFP-B, and mRuby-C endows the ternary complexes with lactose-binding affinity that can be tuned by varying the number of galectin-3 domains integrated into the complex from one to three, while maintaining BRET/FRET function. The modular nature of the fusion protein design, the precise control of domain stoichiometry, and the multiplicity afforded by the three-stranded coiled-coil scaffold provides access to a greater range of subunit combinations than what is possible with heterodimeric coiled-coils used previously. We envision that access to this expanded range of co-integrated protein domain diversity will be advantageous for future development of designer supramolecular machines for therapeutic, diagnostic, and biotechnology applications.

Graphical abstract: Heterogeneous protein co-assemblies with tunable functional domain stoichiometry

Supplementary files

Article information

Article type
Paper
Submitted
30 6 2021
Accepted
24 9 2021
First published
24 9 2021

Mol. Syst. Des. Eng., 2022,7, 44-57

Heterogeneous protein co-assemblies with tunable functional domain stoichiometry

S. A. Farhadi, A. Restuccia, A. Sorrentino, A. Cruz-Sánchez and G. A. Hudalla, Mol. Syst. Des. Eng., 2022, 7, 44 DOI: 10.1039/D1ME00083G

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