scholarly journals A fully enzymatic method for site-directed spin labeling of long RNA

2014 ◽  
Vol 42 (15) ◽  
pp. e117-e117 ◽  
Author(s):  
Isabelle Lebars ◽  
Bertrand Vileno ◽  
Sarah Bourbigot ◽  
Philippe Turek ◽  
Philippe Wolff ◽  
...  
Keyword(s):  
Coupling Reaction ◽  
Spin Labeling ◽  
In Vitro Transcription ◽  
Enzymatic Method ◽  
Paramagnetic Resonance ◽  
T4 Rna Ligase ◽  
Labeled Rna ◽  
Site Directed Spin Labeling ◽  
Novel Strategy

Abstract Site-directed spin labeling is emerging as an essential tool to investigate the structural and dynamical features of RNA. We propose here an enzymatic method, which allows the insertion of a paramagnetic center at a specific position in an RNA molecule. The technique is based on a segmental approach using a ligation protocol with T4 RNA ligase 2. One transcribed acceptor RNA is ligated to a donor RNA in which a thio-modified nucleotide is introduced at its 5′-end by in vitro transcription with T7 RNA polymerase. The paramagnetic thiol-specific reagent is subsequently attached to the RNA ligation product. This novel strategy is demonstrated by introducing a paramagnetic probe into the 55 nucleotides long RNA corresponding to K-turn and Specifier Loop domains from the Bacillus subtilis tyrS T-Box leader RNA. The efficiency of the coupling reaction and the quality of the resulting spin-labeled RNA were assessed by Mass Spectrometry, Electron Paramagnetic Resonance (EPR) and Nuclear Magnetic Resonance (NMR). This method enables various combinations of isotopic segmental labeling and spin labeling schemes, a strategy that will be of particular interest to investigate the structural and dynamical properties of large RNA complexes by NMR and EPR spectroscopies.

scholarly journals Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

2019 ◽  
Author(s):  
Pia Widder ◽  
Julian Schuck ◽  
Daniel Summerer ◽  
Malte Drescher
Keyword(s):  
Spin Labeling ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Bioorthogonal Labeling ◽  
In Vitro Measurements ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br>

scholarly journals Combining Site-Directed Spin Labeling in Vivo and In-Cell EPR Distance Determination

2019 ◽  
Author(s):  
Pia Widder ◽  
Julian Schuck ◽  
Daniel Summerer ◽  
Malte Drescher
Keyword(s):  
Spin Labeling ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Determination ◽  
Bioorthogonal Labeling ◽  
In Vitro Measurements ◽  
Site Directed Spin Labeling ◽  
Double Electron Electron Resonance

Structural studies on proteins directly in their native environment are required for a comprehensive understanding of their function. Electron paramagnetic resonance (EPR) spectroscopy and in particular double electron-electron resonance (DEER) distance determination are suited to investigate spin-labeled proteins directly in the cell. The combination of intracellular bioorthogonal labeling with in-cell DEER measurements does not require additional purification or delivery steps of spin-labeled protein to the cells. In this study, we express eGFP in E.coli and use copper-catalyzed azide-alkyne cycloaddition (CuAAC) for the site-directed spin labeling of the protein in vivo, followed by in-cell EPR distance determination. Inter-spin distance measurements of spin-labeled eGFP agree with in vitro measurements and calculations based on the rotamer library of the spin label.<br>

scholarly journals Effects of Fe2+/Fe3+ Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies

2020 ◽  
Vol 21 (24) ◽  
pp. 9619
Author(s):  
Davide Doni ◽  
Leonardo Passerini ◽  
Gérard Audran ◽  
Sylvain R. A. Marque ◽  
Marvin Schulz ◽  
...  
Keyword(s):  
Ferrous Iron ◽  
Iron Homeostasis ◽  
Spin Labeling ◽  
Iron Binding ◽  
Paramagnetic Resonance ◽  
Binding Effect ◽  
Relevant Role ◽  
Long Time ◽  
Site Directed Spin Labeling

Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

scholarly journals Posttranscriptional Site-Directed Spin Labeling of Large RNAs with an Unnatural Base Pair System Under Non-Denaturing Conditions

2020 ◽  
Author(s):  
Yan Wang ◽  
Venkatesan Kathiresan ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Base Pair ◽  
Continuous Wave ◽  
Double Resonance ◽  
Rnase P ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
And Function

<div> <p>Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy remains challenging up-to-date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs under non-denaturing conditions using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3<sup>CO</sup>TP) is synthesized and site-specifically incorporated into large RNAs by <i>in vitro</i> transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy using a 419-nucleotide Ribonuclease P (RNase P) RNA from Bacillus <i>stearothermophilus. </i>The effects of site-directed UBP incorporation and subsequent spin labeling to global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, and enzymatic assay. Continuous-wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees well with the crystal structure. Thus, the labeling strategy as presented overcomes the size constraint of RNA labeling, opening new possibilities for application of EPR spectroscopy in investigating structure and dynamics of large RNA.</p> </div> <br>

scholarly journals Posttranscriptional Site-Directed Spin Labeling of Large RNAs with an Unnatural Base Pair System Under Non-Denaturing Conditions

2020 ◽  
Author(s):  
Yan Wang ◽  
Venkatesan Kathiresan ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Base Pair ◽  
Continuous Wave ◽  
Double Resonance ◽  
Rnase P ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
And Function

<div> <p>Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy remains challenging up-to-date. We here demonstrate an efficient and generally applicable posttranscriptional SDSL method for large RNAs under non-denaturing conditions using an expanded genetic alphabet containing the NaM-TPT3 unnatural base pair (UBP). An alkyne-modified TPT3 ribonucleotide triphosphate (rTPT3<sup>CO</sup>TP) is synthesized and site-specifically incorporated into large RNAs by <i>in vitro</i> transcription, which allows attachment of the azide-containing nitroxide through click chemistry. We validate this strategy using a 419-nucleotide Ribonuclease P (RNase P) RNA from Bacillus <i>stearothermophilus. </i>The effects of site-directed UBP incorporation and subsequent spin labeling to global structure and function of RNase P are marginal as evaluated by Circular Dichroism spectroscopy, Small Angle X-ray Scattering, and enzymatic assay. Continuous-wave EPR analyses reveal that the labeling reaction is efficient and specific, and Pulsed Electron-Electron Double Resonance measurements yield an inter-spin distance distribution that agrees well with the crystal structure. Thus, the labeling strategy as presented overcomes the size constraint of RNA labeling, opening new possibilities for application of EPR spectroscopy in investigating structure and dynamics of large RNA.</p> </div> <br>

scholarly journals Effect of Type I Antifreeze Proteins on the Freezing and Melting Processes of Cryoprotective Solutions Studied by Site-Directed Spin Labeling Technique

Crystals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 352 ◽  
Author(s):  
Adiel F. Perez ◽  
Kyle R. Taing ◽  
Justin C. Quon ◽  
Antonia Flores ◽  
Yong Ba
Keyword(s):  
Freezing Point ◽  
Antifreeze Proteins ◽  
Active Role ◽  
Spin Labeling ◽  
Ice Crystals ◽  
Freezing Injury ◽  
Type I ◽  
Paramagnetic Resonance ◽  
Potential Applications ◽  
Site Directed Spin Labeling

Antifreeze proteins (AFPs) protect organisms living in subzero environments from freezing injury, which render them potential applications for cryopreservation of living cells, organs, and tissues. Cryoprotective agents (CPAs), such as glycerol and propylene glycol, have been used as ingredients to treat cellular tissues and organs to prevent ice crystal’s formation at low temperatures. To assess AFP’s function in CPA solutions, we have the applied site-directed spin labeling technique to a Type I AFP. A two-step process to prevent bulk freezing of the CPA solutions was observed by the cryo-photo microscopy, i.e., (1) thermodynamic freezing point depression by the CPAs; and (2) inhibition to the growth of seed ice crystals by the AFP. Electron paramagnetic resonance (EPR) experiments were also carried out from room temperature to 97 K, and vice versa. The EPR results indicate that the spin labeled AFP bound to ice surfaces, and inhibit the growths of ice through the bulk freezing processes in the CPA solutions. The ice-surface bound AFP in the frozen matrices could also prevent the formation of large ice crystals during the melting processes of the solutions. Our study illustrates that AFPs can play an active role in CPA solutions for cryopreservation applications.

scholarly journals Posttranscriptional site-directed spin labeling of large RNAs with an unnatural base pair system under non-denaturing conditions

2020 ◽  
Vol 11 (35) ◽  
pp. 9655-9664
Author(s):  
Yan Wang ◽  
Venkatesan Kathiresan ◽  
Yaoyi Chen ◽  
Yanping Hu ◽  
Wei Jiang ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Epr Spectroscopy ◽  
Base Pair ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

Site-directed spin labeling (SDSL) of large RNAs for electron paramagnetic resonance (EPR) spectroscopy has remained challenging to date.

scholarly journals Characterization of the Linker 2 Region in Human Vimentin Using Site-Directed Spin Labeling and Electron Paramagnetic Resonance†

Biochemistry ◽  
2006 ◽  
Vol 45 (39) ◽  
pp. 11737-11743 ◽  
Author(s):  
John F. Hess ◽  
Madhu S. Budamagunta ◽  
Rebecca L. Shipman ◽  
Paul G. FitzGerald ◽  
John C. Voss
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic
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