Prolines in linkers of multi-domain proteins: Intrinsic regulators of misfolding and aggregation

Proline is a chemically unique amino acid that influences protein structure and slows peptide-bond formation during translation. While its enrichment in intrinsically disordered regions is well known, the functional importance of its organization along protein sequences remains unclear. Here, we performed a proteome-wide analysis of inter-domain linkers in human proteins to investigate how proline organization relates to domain architecture and folding demands. We find that clustered and consecutive prolines are enriched in linkers following topologically complex domains that require the formation of long-range contacts. This suggests that proline clusters may act as intrinsic sequence-encoded pauses during translation, helping coordinate co-translational folding. We further show that linkers with enriched proline clusters frequently flank aggregation-prone domains and that proteins containing such linkers tend to have longer cellular half-lives. Together, our results demonstrate that the organization of proline residues, beyond their overall abundance, is an important determinant of protein folding, aggregation, and stability, revealing a general mechanism by which amino acid sequences regulate protein behavior.