Liquid–liquid
phase separation (LLPS) of heterogeneous 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.