Issue 8, 2024

Supramolecular assembly of multifunctional protein gels via an N-glycosylation consensus sequence fusion domain

Abstract

Polypeptide fusion tags that can direct the assembly of folded proteins into supramolecular networks are attractive for creating functional biomaterials. A practical challenge is identifying polypeptide sequences that form supramolecular networks in response to specific user-controlled stimuli, which is advantageous for producing polypeptide–protein fusions using cell-based expression hosts. Here, we report an N-glycosylation tag, (GGGSGGGSGGNWTT)10 or “NGT,” that assembles into a supramolecular network at reduced temperatures when fused to a folded protein. For example, NGT fused to superfolder green fluorescent protein (NGTsfGFP) formed materials that emitted green fluorescence in blue light, while NGT fused to NanoLuc luciferase (NGTnL) formed materials that emitted blue light in the presence of the chemical substrate furimazine. Oscillatory rheology established the materials as weak viscoelastic gels that can undergo shear-thinning and self-healing. Gel formation could be disrupted by mutating the asparagines in NGT to glutamines, introducing a chaotropic agent, or modifying the asparagines in NGT with glucose, suggesting a role for hydrogen bonds involving asparagine in supramolecular network formation. A mixture of soluble NGTsfGFP and NGTnL formed a multifunctional gel at reduced temperature that demonstrated bioluminescence resonance energy transfer between the nL and sfGFP domains in the presence of furimazine. Collectively, these data establish NGT as a temperature-responsive polypeptide tag that can be used to create functional biomaterials from soluble fusion proteins synthesized by cell-based hosts.

Graphical abstract: Supramolecular assembly of multifunctional protein gels via an N-glycosylation consensus sequence fusion domain

Supplementary files

Article information

Article type
Paper
Submitted
09 Feb 2024
Accepted
18 May 2024
First published
24 May 2024

Mol. Syst. Des. Eng., 2024,9, 875-884

Supramolecular assembly of multifunctional protein gels via an N-glycosylation consensus sequence fusion domain

E. D. Hill, S. Michel, N. R. Sequeira, B. G. Keselowsky and G. A. Hudalla, Mol. Syst. Des. Eng., 2024, 9, 875 DOI: 10.1039/D4ME00029C

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