Probing unfolded and intrinsically disordered proteins with single-molecule spectroscopy

The functions of proteins have traditionally been linked to their well-defined three-dimensional, folded structures. It is becoming increasingly clear, however, that many proteins perform essential functions without being folded. Single-molecule spectroscopy provides new opportunities for investigating the structure and dynamics of such unfolded or ‘intrinsically disordered’ proteins (IDPs). The combination of single-molecule Förster resonance energy transfer (FRET) with nanosecond correlation spectroscopy, microfluidic mixing, and related methods can be used to probe intra- and intermolecular distance distributions, reconfiguration dynamics, and interactions on a wide range of timescales, and even in heterogeneous environments, including live cells.