scholarly journals Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

2021 ◽  
Author(s):  
Graham Heaven ◽  
Michael A. Hollas ◽  
Lydia Tabernero ◽  
Alistair J. Fielding
Keyword(s):  
Spin Label ◽  
Tyrosine Phosphatase ◽  
Spin Labeling ◽  
Selective Labeling ◽  
Structural Investigations ◽  
Knock Out ◽  
Double Electron ◽  
Site Directed Spin Labeling ◽  
Biological Complexes ◽  
Double Electron Electron Resonance

AbstractStructural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies.

scholarly journals Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts

2016 ◽  
Vol 18 (8) ◽  
pp. 5819-5831 ◽  
Author(s):  
Zhanglong Liu ◽  
Thomas M. Casey ◽  
Mandy E. Blackburn ◽  
Xi Huang ◽  
Linh Pham ◽  
...  
Keyword(s):  
Spin Labeling ◽  
Conformational Sampling ◽  
Electron Resonance ◽  
Pulsed Epr ◽  
Double Electron ◽  
Conformational Landscape ◽  
Double Electron Electron Resonance ◽  
Hiv 1

The conformational landscape of HIV-1 protease can be characterized by double electron–electron resonance (DEER) spin-labeling.

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 A Gadolinium Spin Label with Both a Narrow Central Transition and Short Tether for Use in Double Electron Electron Resonance Distance Measurements

2019 ◽  
Vol 58 (5) ◽  
pp. 3015-3025 ◽  
Author(s):  
Anokhi Shah ◽  
Amandine Roux ◽  
Matthieu Starck ◽  
Jackie A. Mosely ◽  
Michael Stevens ◽  
...  
Keyword(s):  
Spin Label ◽  
Distance Measurements ◽  
Electron Resonance ◽  
Central Transition ◽  
Double Electron ◽  
Double Electron Electron Resonance

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 Attaching a spin to a protein -- site-directed spin labeling in structural biology.

2007 ◽  
Vol 54 (2) ◽  
pp. 235-244 ◽  
Author(s):  
Aleksander Czogalla ◽  
Aldona Pieciul ◽  
Adam Jezierski ◽  
Aleksander F Sikorski
Keyword(s):  
Conformational Changes ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Spin Labeling ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Structure And Dynamics ◽  
Site Directed Spin Labeling ◽  
Biological Complexes ◽  
Electron Paramagnetic ◽  
Structure Of Proteins

Site-directed spin labeling and electron paramagnetic resonance spectroscopy have recently experienced an outburst of multiple applications in protein science. Numerous interesting strategies have been introduced for determining the structure of proteins and its conformational changes at the level of the backbone fold. Moreover, considerable technical development in the field makes the technique an attractive approach for the study of structure and dynamics of membrane proteins and large biological complexes at physiological conditions. This review focuses on a brief description of site-directed spin labeling-derived techniques in the context of their recent applications.

scholarly journals Methodology for rigorous modeling of protein conformational changes by Rosetta using DEER Distance Restraints

2021 ◽  
Vol 17 (6) ◽  
pp. e1009107
Author(s):  
Diego del Alamo ◽  
Kevin L. Jagessar ◽  
Jens Meiler ◽  
Hassane S. Mchaourab
Keyword(s):  
Conformational Changes ◽  
Spin Label ◽  
Protein Conformational Changes ◽  
Alternative Protein ◽  
Distance Distributions ◽  
Distance Restraints ◽  
Double Electron ◽  
The Time Domain ◽  
Double Electron Electron Resonance ◽  
Experimental Structure

We describe an approach for integrating distance restraints from Double Electron-Electron Resonance (DEER) spectroscopy into Rosetta with the purpose of modeling alternative protein conformations from an initial experimental structure. Fundamental to this approach is a multilateration algorithm that harnesses sets of interconnected spin label pairs to identify optimal rotamer ensembles at each residue that fit the DEER decay in the time domain. Benchmarked relative to data analysis packages, the algorithm yields comparable distance distributions with the advantage that fitting the DEER decay and rotamer ensemble optimization are coupled. We demonstrate this approach by modeling the protonation-dependent transition of the multidrug transporter PfMATE to an inward facing conformation with a deviation to the experimental structure of less than 2Å Cα RMSD. By decreasing spin label rotamer entropy, this approach engenders more accurate Rosetta models that are also more closely clustered, thus setting the stage for more robust modeling of protein conformational changes.

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