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ABSTRACT Biomolecular condensates represent unique microenvironments that organize intracellular biology and promote biochemical reactions. However, the biomolecular interactions driving condensate phase separation are often weak, transient, and heterogeneous. Investigating the structural biology and chemical properties of condensate interiors has therefore proven experimentally challenging, often requiring the use of perturbative probes. To overcome this challenge, we combine label-free optical scattering and vibrational spectroscopy approaches spanning ultraviolet, visible, mid-infrared, and terahertz wavelengths with deep-learning-based ensemble prediction of intrinsically disordered protein conformations. Our experimental and computational results reveal that the intrinsically disordered N-terminal domain of the RNA Deadbox helicase 4 (DDX4) deviates from random coil behavior and undergoes chain collapse that correlates with phase separation, which leads to lower dielectric and reduced water content inside condensates. Our data support a model of DDX4 phase separation whereby chain collapse, reduced dielectric, and water release enhance the strength of multivalent protein-protein interactions within condensates, driving condensate growth and phase separation through positive feedback. Our study addresses the critical driving forces of biomolecular phase separation across a range of length scales, providing quantitative insights into protein-protein/protein-solvent interactions and the chemical properties of condensate interiors.
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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:
- 10414746
- 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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- Free Publicly Accessible Full Text
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Accepted Manuscript
- Journal Article:
- https://doi.org/10.1038/s41467-023-37563-0
