EPR of site-directed spin-labeled proteins: A powerful tool to study structural flexibility

García-Rubio, Inés

doi:10.1016/j.abb.2020.108323

Cited by 19 publications

(21 citation statements)

References 98 publications

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“…Electron Paramagnetic Resonance (EPR) can provide valuable information about the oxidation state of an atom or atoms within a (macro)molecule and its environment by using unpaired electrons as probes. The technique has been used to study various paramagnetic species [1,2] such as organic radicals, [3][4][5] proteins, [6][7][8][9] polymers [5,10] and transition-metal complexes. [11,12] Although EPR spectrometers operating at up to 263 GHz are now commercially available, X-band (~9 GHz) equipment is still the most widely used.…”

Section: Introductionmentioning

confidence: 99%

A Versatile In‐Situ Electron Paramagnetic Resonance Spectro‐electrochemical Approach for Electrocatalyst Research

et al. 2020

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Empirical electrocatalyst research generally consists of the synthesis and experimental characterization of catalysts and the analysis of electrolysis products by conventional analytical techniques. In‐situ electron paramagnetic resonance spectro‐electrochemistry provides an evidence‐based in‐depth understanding of the formed intermediates and the reaction mechanism enabling the desired tuning of electrocatalysts. The use of this technique has been underexploited because of the opposite requirements they impose on the conventional setup. In this work, a versatile electrode with commercially available indium tin oxide on polyethylene terephthalate (PET) was constructed for the first time which can fit inside commonly used flat cells. It allows reproducible electrodeposition of catalytic material combined with sensitive radical detection, owing to its large surface area and minimal disruption to the resonator's Q‐factor. Moreover, with a resistivity of 8–10 Ω sq−1, the surface potential of the thin semiconductor electrode within the resonator was well‐controlled, allowing targeted radical production.

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Section: Introductionmentioning

confidence: 99%

A Versatile In‐Situ Electron Paramagnetic Resonance Spectro‐electrochemical Approach for Electrocatalyst Research

et al. 2020

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“…To assess the potential propensity of NUPR1 to change local conformation in the presence of crowders, the EPR spectra of A2C-MTSL and N72C-MTSL samples were obtained in aqueous solution ( Figures 2 , 3 , red line) and in the presence of the crowding agents, Ficoll-70 and Dextran-40 ( Figures 2 , 3 , black line). The EPR spectrum of A2C-MTSL in aqueous solution showed a typical spectral line shape of a disordered protein ( Longhi et al, 2011 ; Bonucci et al, 2020 ; García-Rubio, 2020 ). Simulation of the spectrum ( Supplementary Figure S7 ) revealed that it is composed of a single sharp component with a fast correlation time value of 0.12 ns ( Table 1 ; Figure 2A ).…”

Section: Resultsmentioning

confidence: 99%

“…A high degree of protein flexibility yields sharp EPR signals (τ c < 1 ns), whereas broader EPR spectra (τ c > 1 ns) are usually recorded when more compact conformations are present. For these reasons, SDSL-EPR spectroscopy with nitroxide spin label has emerged as a valid approach for the characterization of various IDPs and to check possible local disorder-to-order transitions ( Kavalenka et al, 2010 ; Longhi et al, 2011 ; Palombo et al, 2017 ; Bonucci et al, 2020 ; García-Rubio, 2020 ).…”

Section: Resultsmentioning

confidence: 99%

“…The use of crowders yielded an increase in solution viscosity (Supplementary Figure S5) which can strongly affect the tumbling motion of the nitroxide label attached to the protein, as it does for the whole protein (see Far-UV CD). Since the EPR spectral line shapes depend also on the dynamics of spin labelled side chains (Bordignon, 2017;García-Rubio, 2020;Klare, 2013), we decided to acquire EPR spectra of A2C-MTSL and N72C-MTSL in the presence of sucrose (at 30% v/v) to test the effect of viscosity (Supplementary Figure S7 and Supplementary Figure S8). Sucrose is generally considered as an osmolyte with a behavior different from typical crowders (such as Ficoll-70 or Dextran-40) (Fleissner et al, 2009).…”

Section: Eprmentioning

confidence: 99%

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Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Bonucci

Palomino‐Schätzlein

Molina

et al. 2021

Front. Mol. Biosci.

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The intracellular environment is crowded with macromolecules, including sugars, proteins and nucleic acids. In the cytoplasm, crowding effects are capable of excluding up to 40% of the volume available to any macromolecule when compared to dilute conditions. NUPR1 is an intrinsically disordered protein (IDP) involved in cell-cycle regulation, stress-cell response, apoptosis processes, DNA binding and repair, chromatin remodeling and transcription. Simulations of molecular crowding predict that IDPs can adopt compact states, as well as more extended conformations under crowding conditions. In this work, we analyzed the conformation and dynamics of NUPR1 in the presence of two synthetic polymers, Ficoll-70 and Dextran-40, which mimic crowding effects in the cells, at two different concentrations (50 and 150 mg/ml). The study was carried out by using a multi-spectroscopic approach, including: site-directed spin labelling electron paramagnetic resonance spectroscopy (SDSL-EPR), nuclear magnetic resonance spectroscopy (NMR), circular dichroism (CD), small angle X-ray scattering (SAXS) and dynamic light scattering (DLS). SDSL-EPR spectra of two spin-labelled mutants indicate that there was binding with the crowders and that the local dynamics of the C and N termini of NUPR1 were partially affected by the crowders. However, the overall disordered nature of NUPR1 did not change substantially in the presence of the crowders, as shown by circular dichroism CD and NMR, and further confirmed by EPR. The changes in the dynamics of the paramagnetic probes appear to be related to preferred local conformations and thus crowding agents partially affect some specific regions, further pinpointing that NUPR1 flexibility has a key physiological role in its activity.

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“…The technique provides complementary information to that of NMR concerning the different correlation times explored by proteins and IDPs. García-Rubio [18] describes how the introduction of several probes in different positions allows one to obtain reliable distributions of distances in an IDP and how its flexibility and the population of conformers is shifted upon binding to its natural partners. The review can be also used as a general introduction for those hoping to use the benefits of the technique for well-folded proteins.…”

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confidence: 99%

Intrinsically disordered proteins in biology: One for all, all for one

Yruela

Neira

2020

Archives of Biochemistry and Biophysics

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EPR of site-directed spin-labeled proteins: A powerful tool to study structural flexibility

Cited by 19 publications

References 98 publications

A Versatile In‐Situ Electron Paramagnetic Resonance Spectro‐electrochemical Approach for Electrocatalyst Research

A Versatile In‐Situ Electron Paramagnetic Resonance Spectro‐electrochemical Approach for Electrocatalyst Research

Crowding Effects on the Structure and Dynamics of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1

Intrinsically disordered proteins in biology: One for all, all for one

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