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.
Highlights
Characterizing dynamics of macromolecules is a complex task requiring the use of appropriate techniques
Taken together the results demonstrated that phosphorylated maleimido proxyl (PP) is able to monitor from subtle to larger structural transitions, as efficiently as the classical spin label
The partner protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) induced the cleavage of the disulfide bridge between the cysteine and the label, resulting in the full release of the label. This result showed the existence of a transitory interaction between both proteins and we proposed a mechanism based on a thioldisulfide exchange reaction involving cysteines C21 and C291 of the C. reinhardtii GAPDH (Erales et al, 2009)
Summary
Characterizing dynamics of macromolecules is a complex task requiring the use of appropriate techniques. Among the various techniques able to give access to dynamic properties of biomolecules is Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR), a technique that was pioneered about 20 years ago by (Hubbell et al, 1996). The development of pulse EPR, in particular Double Electron Electron Resonance (DEER) techniques allowed the measurement of distances between spin labeled sites in the range of 1.8–6.0 nm (Pannier et al, 2000), covering a wide range of interest for the study of large conformational transitions and biomolecule associations. Excellent reviews describing all of these approaches, as well as applications of SDSL-EPR on proteins, have been recently published (Klare and Steinhoff, 2009; Bordignon, 2011; Mchaourab et al, 2011; Drescher, 2012; Hubbell et al, 2013). The present contribution will focus on dynamic analyses of extremely flexible biological systems and recent synthesis of new spin labels designed to enlarge the potentialities of the technique
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