Nitroxide spin labels: fabulous spy spins for biostructural EPR applications

Abstract

Characterizing proteins in action requires appropriate biophysical techniques sensitive to protein motions. One of the technique dedicated to monitor protein dynamics is Site-Directed Spin Labelling combined with EPR spectroscopy (SDSL-EPR). The main purpose of this chapter is to describe and illustrate the different strategies based on the use of nitroxide spin labels either as reporters or as a means to measure inter-label distances. The complementarity of these different approaches to answer biological questions will be addressed. The objective is also to give non-specialist readers an overview of the recent developments in the field of SDSL-EPR dedicated to the study of protein dynamics. A particular emphasis will be devoted to describe the design and application of new nitroxide spin labels that allow overcoming the limitations of the classical ones.

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

“…SDSL-EPR involves the grafting of a paramagnetic label, generally a thiol-specific nitroxide, on the protein of interest and the determination of the dynamic properties of the attached nitroxide by continuous wave (CW)-EPR spectroscopy [40,41]. Changes in the nitroxide spectrum are thoroughly related to the mobility of the nitroxide side-chain and to the local backbone motion, which can thus be used to follow protein structural changes and to reveal interaction sites in complexes in solution and at room temperature [40,[42][43][44][45]. Distance distributions between two spin labels can be measured by pulsed double electron-electron resonance (DEER) techniques relying on their dipole-dipole coupling [46,47].…”

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…[4][5][6] Among the PDS techniques, double electron-electron resonance (DEER), also known as pulsed electron double resonance (PELDOR), is the most frequently used due to its robustness. [12][13][14] The most commonly used spin label is (1-oxyl-2,2,5,5-tetra-methylpyrroline-3-methyl)-methanethiosulfonate (MTSSL), which specifically reacts with the thiol group of cysteine residues. [10,11] Conventionally, PDS measurements are performed between two nitroxide spin labels, attached to proteins by site-directed spin labelling (SDSL) of a cysteine residue or of a non-native amino acid, which has been genetically encoded.…”

Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label

“…When grafted on proteins, nitroxide spin labels display EPR spectra whose shape reflects the spin label mobility that is related to local environmental constraints as well as global motional tumbling of the protein [19]. Simulation of the EPR spectral shapes allows determining the dynamic parameter tc (i.e.…”

“…[17,18] In particular, among these techniques, Site-directed Spin Labeling coupled to Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) has emerged as an efficient methodology to monitor protein structure and dynamics. [13,[19][20][21][22][23] In the recent years, numerous chemical reactions have been developed to achieve selective conjugation under mild conditions to maintain protein structure and function. [24][25][26][27] Many of these reactions were developed to target nucleophilic amino acids such as cysteine (C) or lysine (K) residues.…”

“…They are characteristic of labels of restricted mobilities arising from their micro-environments. [19] The presence of several components, even in the case of mutants bearing a single nitroxide label (C60YC165* & C60*C165H) possibly arises from two different rotameric states of the spin labels rather than two different environments encountered by each individual spin label, as already reported in the literature [43,44]. In order to get further insight into the conformation of the C-terminal part, it is therefore necessary to use advanced techniques, e.g.…”