Exploring intrinsically disordered proteins using site-directed spin labeling electron paramagnetic resonance spectroscopy

Abstract

Proteins are highly variable biological systems, not only in their structures but also in their dynamics. The most extreme example of dynamics is encountered within the family of Intrinsically Disordered Proteins (IDPs), which are proteins lacking a well-defined 3D structure under physiological conditions. Among the biophysical techniques well-suited to study such highly flexible proteins, Site-Directed Spin Labeling combined with EPR spectroscopy (SDSL-EPR) is one of the most powerful, being able to reveal, at the residue level, structural transitions such as folding events. SDSL-EPR is based on selective grafting of a paramagnetic label on the protein under study and is limited neither by the size nor by the complexity of the system. The objective of this mini-review is to describe the basic strategy of SDSL-EPR and to illustrate how it can be successfully applied to characterize the structural behavior of IDPs. Recent developments aimed at enlarging the panoply of SDSL-EPR approaches are presented in particular newly synthesized spin labels that allow the limitations of the classical ones to be overcome. The potentialities of these new spin labels will be demonstrated on different examples of IDPs.

Dynamic Protein Interaction Networks and New Structural Paradigms in Signaling

“…It also reinforces the need for better descriptions of the structural ensembles sampled by IDPs/IDRs in isolated and complexed states, which are extremely challenging because of under-representative sampling of the conformer pool, the under-determined nature of the problem, and the need for more accurate ways to calculate experimental data from structural models. Incorporation of distance information from fluorescence-based single-molecule methods 429 or electron paramagnetic resonance (EPR) 430 together with NMR and SAXS data into ensemble calculations may provide additional insights into the conformational properties of the disordered ensemble. Generation and statistical analysis of several different ensembles can also help to yield a final ensemble that is most consistent with the experimental data and to estimate the accuracy of the final ensemble.…”

Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

“…For instance, CW spectra at W-and D-bands provide an enhanced capability to detect sub-nanosecond correlation times [43]. This is particularly suitable for studying IDPs, in which the time scale normally falls into the 0.1 to 2 ns region [37,38,[44][45][46][47][48][69][70][71][72].…”

In‐Cell EPR: Progress towards Structural Studies Inside Cells

“…By far, the largest family of spin labels in SDSL‐EPR spectroscopy are nitroxide radicals, with gadolinium(III) complexes and trityl radicals being interesting alternatives. One of the main advantages of nitroxide spin labels is their high sensitivity to the local environment, which, in the SDSL‐EPR approach, is used to obtain structural and dynamic information on the biomolecule on which the nitroxide is attached …”

“…The consecutive study of the EPR spectra and their parameters provides detailed information on the local mobility of the grafted label. Spin label dynamics is related to local structural properties of the protein under investigation and can thus be used to follow protein's structural changes and to detect interaction sites in complexes or monitor folding events . This kind of EPR studies is conducted in a continuous wave mode (cw‐based EPR) and is carried out in solution.…”