Probing Local Structures of an Intrinsically Disordered Protein Through Single-Molecule Force Stretching Experiments

Understanding the various roles of intrinsically disordered proteins (IDPs) requires having detailed knowledge of their disordered conformations, which can be an experimental challenge. Here, using single-molecule magnetic tweezers, we probe the conformation of a polypeptide construct of the neurofilament subunit protein’s disordered tail by looking at its elastic properties. When stretched at subpicoNewton forces, the IDP behaves nearly like an ideal chain – which was surprising given its high net charge – suggesting local structures within the protein. In guanidinium, these local structures are unfolded as the protein extends in length and behaves as a swollen random coil. We observe gradual extension and swelling with increasing guanidinium concentration, indicating partial unfolding of these structures. Comparing this to the elastic response in other solvent conditions, such as salt and nonionic denaturant, reveals the nature of these local structures. In addition, applying higher forces (<100pN) provides more proof of these local structures by mechanically disrupting them. Overall, we show complex elastic behaviors that reveal local structures in an IDP, and we demonstrate a framework to study the conformations of IDPs through force stretching experiments.