Intrinsically disordered proteins (IDPs) are now widely recognized as playing critical roles in a broad range of cellular functions as well as being implicated in diverse diseases. Their lack of stable secondary structure and tertiary interactions, coupled with their sensitivity to measurement conditions, stymies many traditional structural biology approaches. Single-molecule Förster resonance energy transfer (smFRET) is now widely used to characterize the physicochemical properties of these proteins in isolation and is being increasingly applied to more complex assemblies and experimental environments. This review provides an overview of confocal diffusion-based smFRET as an experimental tool, including descriptions of instrumentation, data analysis, and protein labeling. Recent papers are discussed that illustrate the unique capability of smFRET to provide insight into aggregation-prone IDPs, protein-protein interactions involving IDPs, and IDPs in complex experimental milieus.
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