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Research article2023Peer reviewedOpen access

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures

Sahin, Cagla; Motso, Aikaterini; Gu, Xinyu; Feyrer, Hannes; Lama, Dilraj; Arndt, Tina; Rising, Anna; Gese, Genis Valentin; Haellberg, B. Martin; Marklund, Erik. G.; Schafer, Nicholas P.; Petzold, Katja; Teilum, Kaare; Wolynes, Peter G.; Landreh, Michael

Abstract

Liquid-liquid phase separation (LLPS) of hetero-geneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

Published in

Journal of the American Chemical Society
2023, Volume: 145, number: 19, pages: 10659-10668 Publisher: AMER CHEMICAL SOC

    Sustainable Development Goals

    SDG3 Good health and well-being

    UKÄ Subject classification

    Physical Chemistry
    Biochemistry and Molecular Biology

    Publication identifier

    DOI: https://doi.org/10.1021/jacs.3c00932

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/122371