Intrinsically Disordered Regions Promote Protein Refoldability and Facilitate Retrieval from Biomolecular Condensates–Peptide Quantifications

Many eukaryotic proteins contain intrinsically disordered regions (IDRs) that intersperse globular folded domains, in contrast with bacterial proteins which are typically highly globular. Recent years have seen great progress in identifying biological functions associated with these elusive protein sequence: in specific cases, they mediate liquid- liquid phase separation, perform molecular recognition, or act as sensors to changes in the environment. Nevertheless, only a small number of IDRs have annotated functions despite their presence in 64% of yeast proteins, stimulating some to question what ‘general purpose’ they may serve. Here, by interrogating the refoldability of two fungal proteomes (Saccharomyces cerevisiae and Neurosporra crassa), we show that IDRs render their host proteins more refoldable from the denatured state, allowing them to cohere more closely to Anfinsen’s thermodynamic hypothesis. The data provide an exceptionally clear picture of which biophysical and topological characteristics enable refoldability. Moreover, we find that almost all yeast proteins that partition into stress granules during heat shock are refoldable, a finding that holds for other condensates such as P-bodies and the nucleolus. Finally, we find that the Hsp104 unfoldase is the principal actor in mediating disassembly of heat stress granules and that the efficiency with which condensed proteins are returned to the soluble phase is also well explained by refoldability. Hence, these studies establish spontaneous refoldability as an adaptive trait that endows proteins with the capacity to reform their native soluble structures following their extraction from condensates. Altogether, our results provide an intuitive model for the function of IDRs in many multidomain proteins and clarifies their relationship to the phenomenon of biomolecular condensation.

This dataset provides peptide quantifications (and their respective P-values) from three separate types of experiments used to support the claims in this study.

1. Peptide quantifications from global refolding reactions, assessed with limited-proteolysis mass spectrometry (LiP-MS), carried out on two fungal organisms (S. cerevisiae [yeast] & N. crassa), at three refolding times, repeated on three separate iterations (for yeast).

2. Peptide quantifications from LiP-MS experiments conducted on yeast extracts during heat shock or recovery from heat shock

3. Annotations for peptides in #1 that are associated with linker regions between folded domains.