Abstract
Intrinsically disordered proteins (IDPs) are often modeled using ideas from polymer physics that suggest they smoothly explore all corners of configuration space. Experimental verification of this random, dynamic behavior is difficult as random fluctuations of IDPs cannot be synchronized across an ensemble. Single molecule fluorescence (or Förster) resonance energy transfer (smFRET) is one of the few approaches that are sensitive to transient populations of sub-states within molecular ensembles. In some implementations, smFRET has sufficient time resolution to resolve transitions in IDP behaviors. Here we present experimental issues to consider when applying smFRET to study IDP configuration. We illustrate the power of applying smFRET to IDPs by discussing two cases in the literature of protein systems for which smFRET has successfully reported phosphorylation-induced modification (but not elimination) of the disordered properties that have been connected to impacts on the related biological function. The examples we discuss, PAGE4 and a disordered segment of the GluN2B subunit of the NMDA receptor, illustrate the great potential of smFRET to inform how IDP function can be regulated by controlling the detailed ensemble of disordered states within biological networks.