Label-free optical observation of disordered-to-ordered transitions in single intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) and structured proteins with intrinsically disordered regions (IDRs) lack a definitive tertiary structure and contribute to the onset of diseases such as Alzheimer's and cancer. To date, experimental observation of single, label-free IDPs/IDRs poses a significant challenge due to their structural heterogeneity, limiting ensemble techniques from fully capturing their properties, whilst single-molecule measurements require site-specific modifications or non-physiological conditions, perturbing their native biophysics. Here, we demonstrate the first experimental observation of unmodified IDP/IDR conformational dynamics at the single-molecule level, achieved by optical trapping and investigation of individual IDPs/IDRs using nanoaperture optical tweezers. Our results reveal that IDPs/IDRs exhibit significantly larger conformational variations compared to globular proteins of similar size. 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 the binding of the N-terminal region of the Src-associated protein in mitosis of 68 kDa (Sam68) to G8.5 RNA. These findings present nanoaperture optical tweezers as a powerful approach to advance our understanding of IDPs/IDRs and further decode their roles in associated diseases.