Liquid Droplet Aging and Seeded Fibril Formation of the Cytotoxic Granule Associated RNA Binding Protein TIA1 Low Complexity Domain

[Image: see text] Protein domains biased toward a few amino acid types are vital for the formation of biomolecular condensates in living cells. These membraneless compartments are formed by molecules exhibiting a range of molecular motions and structural order. Missense mutations increase condensate persistence lifetimes or structural order, properties that are thought to underlie pathological protein aggregation. In the context of stress granules associated with neurodegenerative diseases, this process involves the rigidification of protein liquid droplets into β-strand rich protein fibrils. Here, we characterize the molecular mechanism underlying the rigidification of liquid droplets for the low complexity domain of the Cytotoxic granule associated RNA binding protein TIA1 (TIA1) stress granule protein and the influence of a disease mutation linked to neurodegenerative diseases. A seeding procedure and solid state nuclear magnetic resonance measurements show that the low complexity domain converges on a β-strand rich fibril conformation composed of 21% of the sequence. Additional solid state nuclear magnetic resonance measurements and difference spectroscopy show that aged liquid droplets of wild type and a proline-to-leucine mutant low complexity domain are composed of fibril assemblies that are conformationally heterogeneous and structurally distinct from the seeded fibril preparation. Regarding low complexity domains, our data support the functional template-driven formation of conformationally homogeneous structures, that rigidification of liquid droplets into conformationally heterogenous structures promotes pathological interactions, and that the effect of disease mutations is more nuanced than increasing thermodynamic stability or increasing β-strand structure content.