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bioRxiv · 10.1101/2023.05.23.541917

Single-Molecule FRET Illuminates Structural Subpopulations and Dissects Crucial Molecular Events During Phase Separation of a Prion-Like Low Complexity Domain

Joshi, A.·Walimbe, A.·Avni, A.·Rai, S. K.·Arora, L.·Sarkar, S.·Mukhopadhyay, S.
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Abstract

Biomolecular condensates formed via phase separation of proteins and nucleic acids are thought to be associated with a wide range of cellular functions and dysfunctions. We dissect critical molecular events associated with phase separation of an intrinsically disordered prion-like low-complexity domain of Fused in Sarcoma by performing single-molecule studies that permit us to access the wealth of molecular information that is skewed in conventional ensemble experiments. Our single-molecule FRET experiments reveal the coexistence of two conformationally distinct subpopulations in the monomeric form. Single-droplet single-molecule FRET studies coupled with fluorescence correlation spectroscopy, picosecond time-resolved fluorescence anisotropy, and vibrational Raman spectroscopy indicate that structural unwinding switches intramolecular interactions into intermolecular contacts allowing the formation of a dynamic network within condensates. A disease-related mutation introduces enhanced structural plasticity engendering greater interchain interactions that can accelerate pathological aggregation. Our findings provide key mechanistic underpinnings of sequence-encoded dynamically-controlled structural unzipping resulting in biological phase separation.

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Joshi, A., Walimbe, A., Avni, A., Rai, S. K., Arora, L., Sarkar, S., Mukhopadhyay, S.. 2023-05-24. Single-Molecule FRET Illuminates Structural Subpopulations and Dissects Crucial Molecular Events During Phase Separation of a Prion-Like Low Complexity Domain. https://doi.org/10.1101/2023.05.23.541917

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