Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy

BIO-PROTOCOL ◽  
2021 ◽  
Vol 11 (24) ◽  
Author(s):  
Kevin Singewald ◽  
James Wilkinson ◽  
Sunil Saxena
Keyword(s):  
Epr Spectroscopy ◽  
Continuous Wave ◽  
Spin Labeling ◽  
Site Directed Spin Labeling

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 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.

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

2007 ◽  
Vol 37 (4) ◽  
pp. 483-493 ◽  
Author(s):  
Marcin Sarewicz ◽  
Sebastian Szytuła ◽  
Małgorzata Dutka ◽  
Artur Osyczka ◽  
Wojciech Froncisz
Keyword(s):  
Protein Interaction ◽  
Epr Spectroscopy ◽  
Spin Labeling ◽  
Binding Parameters ◽  
Protein Protein Interaction ◽  
Site Directed Spin Labeling ◽  
A Site

Site-directed spin labeling EPR spectroscopy in protein research

2013 ◽  
Vol 394 (10) ◽  
pp. 1281-1300 ◽  
Author(s):  
Johann P. Klare
Keyword(s):  
Nucleic Acids ◽  
Epr Spectroscopy ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Spin Labeling ◽  
Paramagnetic Resonance ◽  
Protein Research ◽  
Site Directed Spin Labeling ◽  
Electron Paramagnetic ◽  
Natronomonas Pharaonis

Abstract Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author’s laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

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