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Intrinsically disordered regions (IDRs) in proteins are often targets of combinatorial posttranslational modifications, which serve to regulate protein structure and function. Emerging evidence suggests that the N-terminal tails of G protein γ subunits, which are essential components of heterotrimeric G proteins, are intrinsically disordered, phosphorylation-dependent determinants of G protein signaling. Here, we found that the yeast Gγ subunit Ste18 underwent combinatorial, multisite phosphorylation events within its N-terminal IDR. G protein–coupled receptor (GPCR) activation and osmotic stress induced phosphorylation at Ser7, whereas glucose and acid stress induced phosphorylation at Ser3, which was a quantitative indicator of intracellular pH. Each site was phosphorylated by a distinct set of kinases, and phosphorylation of one site affected phosphorylation of the other, as determined through exposure to serial stimuli and through phosphosite mutagenesis. Last, we showed that phosphorylation resulted in changes in IDR structure and that different combinations of phosphorylation events modulated the activation rate and amplitude of the downstream mitogen-activated protein kinase Fus3. These data place Gγ subunits among intrinsically disordered proteins that undergo combinatorial posttranslational modifications that govern signaling pathway output.
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Bi-terminal fusion of intrinsically-disordered mussel foot protein fragments boosts mechanical strength for protein fibers
Abstract Microbially-synthesized protein-based materials are attractive replacements for petroleum-derived synthetic polymers. However, the high molecular weight, high repetitiveness, and highly-biased amino acid composition of high-performance protein-based materials have restricted their production and widespread use. Here we present a general strategy for enhancing both strength and toughness of low-molecular-weight protein-based materials by fusing intrinsically-disordered mussel foot protein fragments to their termini, thereby promoting end-to-end protein-protein interactions. We demonstrate that fibers of a ~60 kDa bi-terminally fused amyloid-silk protein exhibit ultimate tensile strength up to 481 ± 31 MPa and toughness of 179 ± 39 MJ*m−3, while achieving a high titer of 8.0 ± 0.70 g/L by bioreactor production. We show that bi-terminal fusion of Mfp5 fragments significantly enhances the alignment of β-nanocrystals, and intermolecular interactions are promoted by cation-π and π-π interactions between terminal fragments. Our approach highlights the advantage of self-interacting intrinsically-disordered proteins in enhancing material mechanical properties and can be applied to a wide range of protein-based materials.
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- PAR ID:
- 10406921
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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