Single Molecule FRET: A Powerful Tool to Study Intrinsically Disordered Proteins

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.

Features of molecular recognition of intrinsically disordered proteins via coupled folding and binding

“…In recent years, however, large progress has been made from a close collaboration between experimental and computational studies. A spectrum of techniques have been applied to study IDPs/IDRs, providing valuable information on their structures, dynamic properties and binding mechanisms . In parallel, molecular modeling and computer simulations provide atomic pictures of conformation ensembles and binding processes as well as reveal important underlying physical principles …”

“…A spectrum of techniques have been applied to study IDPs/IDRs, providing valuable information on their structures, dynamic properties and binding mechanisms. 2,3,[71][72][73][74][75] In parallel, molecular modeling and computer simulations provide atomic pictures of conformation ensembles and binding processes as well as reveal important underlying physical principles. [76][77][78][79][80][81][82] 3 | RATE CONSTANTS Coupled folding with binding has been suggested to enhance the binding rates of IDPs/IDRs.…”

Expansion of Intrinsically Disordered Proteins Increases the Range of Stability of Liquid–Liquid Phase Separation

“…Experimental advances in single molecule Förster resonance energy transfer (smFRET) have now enabled the direct observation of conformational switching in diluted conditions [ 53 , 54 , 55 ]. However, how conformational switching and the role of IDR conformation impacts the liquid–liquid phase behaviour of IDRs is not fully understood.…”

The Order-Disorder Continuum: Linking Predictions of Protein Structure and Disorder through Molecular Simulation

“…Some techniques also have resolution or timescale constraints (as illustrated in Fig. 1), which can affect the ability to capture disorder on residue length scales or longer timescales 23,24 .…”

“…Single molecule fluorescence techniques such as single molecule Förster resonance (smFRET) have helped to describe protein ensembles by capturing long-range transitions between IDP and IDR configurations 23,46 . While newer methods may contribute accurate characterizations of multiple IDP and IDR conformations, models may Figure 1.…”