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 jun 2021
Accepted
24 sep 2021
First published
24 sep 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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