SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments

2017 ◽  
Vol 55 (8) ◽  
pp. 714-719 ◽  
Author(s):  
E. Etienne ◽  
N. Le Breton ◽  
M. Martinho ◽  
E. Mileo ◽  
V. Belle
Keyword(s):  
User Interface ◽  
Graphical User Interface ◽  
Continuous Wave ◽  
Spin Labeling ◽  
Epr Spectra ◽  
Site Directed Spin Labeling

scholarly journals Probing iso-1-cytochrome c structure by site-directed spin labeling and electron paramagnetic resonance techniques.

1999 ◽  
Vol 46 (4) ◽  
pp. 889-899 ◽  
Author(s):  
J Pyka ◽  
A Osyczka ◽  
B Turyna ◽  
W Blicharski ◽  
W Froncisz
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Cytochrome C ◽  
Spin Label ◽  
Continuous Wave ◽  
Spin Labeling ◽  
Local Dynamic ◽  
Epr Spectrum ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic

A cysteine-specific methanethiosulfonate spin label was introduced into yeast iso-1-cytochrome c at three different positions. The modified forms of cytochrome c included: the wild-type protein labeled at naturally occurring C102, and two mutated proteins, S47C and L85C, labeled at positions 47 and 85, respectively (both S47C and L85C derived from the protein in which C102 had been replaced by threonine). All three spin-labeled protein derivatives were characterized using electron paramagnetic resonance (EPR) techniques. The continuous wave (CW) EPR spectrum of spin label attached to L85C differed from those recorded for spin label attached to C102 or S47C, indicating that spin label at position 85 was more immobilized and exhibited more complex tumbling than spin label at two other positions. The temperature dependence of the CW EPR spectra and CW EPR power saturation revealed further differences of spin-labeled L85C. The results were discussed in terms of application of the site-directed spin labeling technique in probing the local dynamic structure of iso-1-cytochrome c.

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>

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