Direct observation of conformational dynamics in intrinsically disordered proteins at the single-molecule level

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) in structured proteins are integral to many biological processes including neurotransmitter regulation, microtubule regulation, and transcription. IDP/IDRs are heterogenous, existing in a conformational ensemble of various interconnected states without a definitive tertiary structure. The high dynamicity of IDPs/IDRs limits ensemble protein characterisation techniques from capturing their properties, and measurements at the single-molecule level are hampered by the necessity to label the protein or modify its microenvironment, affecting their biophysics. Consequently, our understanding of IDPs/IDRs is limited, translating to a lack of knowledge of their roles in related diseases including Alzheimer’s disease, Parkinson’s disease, and various cancers. This work presents the first experimental observation of unmodified IDP/IDR conformational dynamics in vitro , at the single-molecule level in real time, achieved by trapping individual IDPs/IDRs in a nanoscale volume using nanoaperture optical tweezers. Our results reveal that IDPs/IDRs exhibit significantly larger conformational variations in solution compared to globular proteins of similar size, as expected. We demonstrate that phosphorylation of native tau-441 by glycogen synthase kinase 3-beta (GSK3β-tau) induces compaction and reduced conformational dynamics. We further observed a disorder-to-order transition during binding of an IDR, the N-terminal region of Src-associated protein in mitosis of 68 kDa (Sam68), to G8.5 RNA. The capability of nanoaperture optical tweezers to monitor the dynamic behaviours of single, unmodified IDPs/IDRs provides a powerful approach to advance our understanding of their elusive behaviours and further decode their roles in associated diseases.