Methods for the study of polydisperse ribonucleoprotein complexes

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Abstract

Intrinsic disorder is highly prevalent in the proteome and constitutes an essential feature of many dynamic multiprotein complexes. Flexibility can persist within complexes that are cooperatively held together by spatially distributed weak binding interfaces. Furthermore, the flexibility of constituent macromolecular components can allow for coexisting assemblies with a range of stoichiometries. During the reporting period, we have observed the characteristic features of disordered protein complexes described above in SARS-CoV-2 ribonucleoprotein particles (RNPs), which are formed by the viral nucleocapsid protein (N-protein) and viral RNA. In the viral life cycle, RNPs condense the genomic viral RNA for packing in new virions. RNPs can serve as a model system for complexes of disordered proteins, as they can be assembled in vitro from purified N-protein and different RNA ligands. Dependent on the nucleic acid, six to ten N-protein dimers co-assemble with nucleic acid of several hundred nucleotides in total length. Using the combination of sedimentation velocity analytical ultracentrifugation (SV) and mass photometry (MP) we have characterized the size-distribution of polydisperse RNP mixtures and measured RNP stabilities. This led to the discovery of naturally occurring N-protein mutant species with enhanced protein-protein interfaces and increased RNP stability. Subsequently, we selected a stem-loop RNA ligand that further enhanced complex stability and simultaneously reduced sample polydispersity. Using these in vitro assembled RNP complexes we embarked on structure determination by cryo-electron microscopy. The residual polydispersity in the oligomeric state can be discriminated in image classes with different symmetry. Through this ongoing work we aim at elucidating the detailed architecture of RNPs, and at the same time developing a roadmap for the study of flexible viral multi-protein complexes. Publications: RNP-stabilizing mutants and the methodology combining SV and MP are described in our bioRxiv preprint (10.1101/2025.04.26.650775).