Nitroxide spin labels and EPR spectroscopy: A powerful association for protein dynamics studies

Torricella, Francesco; Pierro, Annalisa; Mileo, Elisabetta; Belle, Valérie; Bonucci, Alessio

doi:10.1016/j.bbapap.2021.140653

Cited by 75 publications

(67 citation statements)

References 99 publications

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“…An interesting example of the last class is represented by the nitroxides family, which are aromatic radicals characterized by a long-lived spin-unpaired electronic ground state: the unpaired electron is localized on the N-O group (Jeschke, 2013;Torricella et al, 2021). For this reason nitroxide radicals are often used in site-directed spin labelling (SDSL; Klare & Steinhoff, 2009) of diamagnetic biomacromolecules (Kugele et al, 2019), which in turn allows the study of the stereochemical structure and dynamical information of biomolecular complexes using electron paramagnetic resonance 1 (EPR; Altenbach et al, 1990;Zerbetto et al, 2007;Torricella et al, 2021). The latter is a spectroscopic technique based on magnetic resonance principles that allows the characterization of systems containing unpaired electrons to determine information on their chemical environment with high resolution.…”

Section: Introductionmentioning

confidence: 99%

A radical approach to radicals

Liu

Biczysko

Moriarty

2022

Acta Cryst Sect D Struct Biol

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Nitroxide radicals are characterized by a long-lived spin-unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site-directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein–ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re-refined by defining its ideal structural parameters based on quantum-chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data.

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Section: Introductionmentioning

confidence: 99%

A radical approach to radicals

Liu

Biczysko

Moriarty

2022

Acta Cryst Sect D Struct Biol

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“…In order to monitor drugs with EPR spectroscopy, spin labeled drugs (SLSA, SLIbf and SLChl) were synthesized using stable Tempo (2,2,6,6‐tetramethylpiperidine‐1‐oxyl) based nitroxide radicals (Figure 1). [20,42,43] This technique has already been used to investigate ligand and drug bindings to albumin proteins [20,43–45] . However, according to our knowledge, spin labeled drug binding to any nanoparticles has not been studied with EPR spectroscopy yet.…”

Section: Introductionmentioning

confidence: 99%

Characterization of Water Solubility and Binding of Spin Labeled Drugs in the Presence of Albumin Nanoparticles and Proteins by Electron Paramagnetic Resonance Spectroscopy

Sozer

Akdoğan

2022

ChemistrySelect

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Electron paramagnetic resonance (EPR) spectroscopy is an advantageous technique to monitor solubility of drugs in an aqueous solution. In the presence of a drug carrier, the bound and unbound drug fractions can be determined in the same sample simultaneously. To enhance the solubility of hydrophobic drugs, a transporter protein of bovine serum albumin (BSA) can be used directly or in the form of nanoparticle. Moreover, a cationic BSA can be used to enhance anionic drug loading. Here, drugs with different water solubility, salicylic acid (high), ibuprofen (low) and chlorambucil (none) were spin labeled and studied with EPR spectroscopy. Remarkably, it has been shown that albumin nanoparticles are much more effective than albumin proteins in dissolving hydrophobic drugs in water. Furthermore, different drug loading methods were compared, and different from other techniques drug release can be monitored directly from the NPs pellet dissolution by EPR spectroscopy.

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“…To complement the NMR data, we resorted to electron paramagnetic resonance (EPR) spectroscopy, another technique suitable to detect dynamics and minor conformational states of proteins in lipid bilayers, but sensitive to longer distances than can be measured by NMR (Sahu & Lorigan, 2020; Torricella et al, 2021). Distances from about 1.8 to 8 nm between paramagnetic spin labels attached to membrane proteins can be measured with pulsed electron double resonance (PELDOR) EPR experiments and can provide insight into non-homogeneous conformational ensembles (Jeschke, 2012).…”

Section: Resultsmentioning

confidence: 99%

“…We made use of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopies for their ability to characterize molecular structure and dynamics as well as minor conformational states on complementary time scales and distance ranges (Mittermaier & Kay, 2009;Torricella et al, 2021). In addition, both techniques can be applied to proteins in lipid bilayer environments i.e., liposomes and lipid nanodiscs (Liang & Tamm, 2016;Sahu & Lorigan, 2020).…”

Section: Conformational Changes Of Fhac In a Lipid Environmentmentioning

confidence: 99%

Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

Sicoli

Konijnenberg

Guérin

et al. 2021

Preprint

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The Two-Partner secretion pathway mediates protein transport across the outer membrane of Gram-negative bacteria. TpsB transporters belong to the Omp85 superfamily, whose members catalyze protein insertion into, or translocation across membranes without external energy sources. They are composed of a transmembrane β barrel preceded by two periplasmic POTRA domains that bind the incoming protein substrate. Here we used an integrative approach combining in vivo assays, mass spectrometry, nuclear magnetic resonance and electron paramagnetic resonance techniques suitable to detect minor states in heterogeneous populations, to explore transient conformers of the TspB transporter FhaC. This revealed substantial, spontaneous conformational changes with a portion of the POTRA2 domain coming close to the lipid bilayer and surface loops. Specifically, the amphipathic β hairpin immediately preceding the first barrel strand can insert into the β barrel. We propose that these motions enlarge the channel and may hoist the substrate into it for secretion. An anchor region at the interface of the β barrel and the POTRA2 domain stabilizes the transporter in the course of secretion. Our data propose a solution to the conundrum how these proteins mediate protein secretion without the need for cofactors, by utilizing intrinsic protein dynamics.

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Nitroxide spin labels and EPR spectroscopy: A powerful association for protein dynamics studies

Cited by 75 publications

References 99 publications

A radical approach to radicals

A radical approach to radicals

Characterization of Water Solubility and Binding of Spin Labeled Drugs in the Presence of Albumin Nanoparticles and Proteins by Electron Paramagnetic Resonance Spectroscopy

Large-scale conformational changes of FhaC provide insights into the two-partner secretion mechanism

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