Abstract
Single molecule fluorescence and nuclear magnetic resonance spectroscopy (NMR) are two very powerful techniques for the analysis of intrinsically disordered proteins (IDPs). Both techniques have individually made major contributions to deciphering the complex properties of IDPs and their interactions, and it has become evident that they can provide very complementary views on the distance-dynamics relationships of IDP systems. We now review the first approaches using both NMR and single molecule fluorescence to decipher the molecular properties of IDPs and their interactions. We shed light on how these two techniques were employed synergistically for multidomain proteins harboring intrinsically disordered linkers, for veritable IDPs, but also for liquid–liquid phase separated systems. Additionally, we provide insights into the first approaches to use single molecule Förster resonance energy transfer (FRET) and NMR for the description of multiconformational models of IDPs.
Highlights
Disordered proteins (IDPs), that is, proteins without stable, threedimensional structures, have gained increasing interest in the recent past as their importance within many biological processes has become evident
We provide an overview on approaches that have been performed on folded proteins containing intrinsically disordered linkers, large intrinsically disordered proteins involved in various biological mechanisms as well as those engaged in liquid–liquid phase separation
The ratio between these two states was shown to be modulated as a function of Y88 and Y74 phosphorylation, depending on the position of the fluorescent label sensing the dynamic motion of the protein chain, in agreement with the release of both phosphorylated sites from Cdk2 as observed by nuclear magnetic resonance spectroscopy (NMR). single molecule Förster Resonance Energy Transfer (smFRET) of p27 labelled with a donor and an acceptor fluorophore for smFRET revealed a very broad population again in agreement with the presence of a minor state in slow exchange with the major state, as determined by photon distribution analysis (PDA) [71]
Summary
Disordered proteins (IDPs), that is, proteins without stable, threedimensional structures, have gained increasing interest in the recent past as their importance within many biological processes has become evident. The calculation of fluorescence correlation curves from single molecule traces (fluorescence correlation spectroscopy, FCS), for example, has made it possible to investigate time scales ranging from picoseconds to several milliseconds in a quantitative way [18,33], thereby addressing time scales in the few microseconds/hundreds nanoseconds regime, which are not directly accessible by NMR [34,35] All these features make single molecule fluorescence and NMR spectroscopy an extremely powerful pair of techniques with which to analyze the conformational dynamics of IDPs. All these features make single molecule fluorescence and NMR spectroscopy an extremely powerful pair of techniques with which to analyze the conformational dynamics of IDPs This realization has led to the first studies combining single molecule fluorescence (in particular smFRET) and NMR for the analysis of IDPs. In this review, we provide an overview on approaches that have been performed on folded proteins containing intrinsically disordered linkers, large intrinsically disordered proteins involved in various biological mechanisms as well as those engaged in liquid–liquid phase separation. Been demonstrated for a describe the conformational landscape of IDPs in a quantitative few examples
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