Posttranscriptional spin labeling of RNA by tetrazine-based cycloaddition

2019 ◽  
Vol 17 (7) ◽  
pp. 1805-1808 ◽  
Author(s):  
Christof Domnick ◽  
Gregor Hagelueken ◽  
Frank Eggert ◽  
Olav Schiemann ◽  
Stephanie Kath-Schorr
Keyword(s):  
Click Chemistry ◽  
Spin Labeling ◽  
Spin Labels ◽  
Distance Distributions ◽  
Nitroxide Spin Labels

Spin labeling of in vitro transcribed RNA by iEDDA click chemistry is demonstrated. This allows the determination of distance distributions between two nitroxide spin labels by PELDOR in a self-complementary RNA duplex.

Pulsed electron–electron double resonance: beyond nanometre distance measurements on biomacromolecules

2011 ◽  
Vol 434 (3) ◽  
pp. 353-363 ◽  
Author(s):  
Gunnar W. Reginsson ◽  
Olav Schiemann
Keyword(s):  
Protein Complexes ◽  
Double Resonance ◽  
Resonance Method ◽  
Spin Labels ◽  
Coupling Mechanism ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Distance Distributions ◽  
Nitroxide Spin Labels ◽  
Amino Acid Radicals

PELDOR (or DEER; pulsed electron–electron double resonance) is an EPR (electron paramagnetic resonance) method that measures via the dipolar electron–electron coupling distances in the nanometre range, currently 1.5–8 nm, with high precision and reliability. Depending on the quality of the data, the error can be as small as 0.1 nm. Beyond mere mean distances, PELDOR yields distance distributions, which provide access to conformational distributions and dynamics. It can also be used to count the number of monomers in a complex and allows determination of the orientations of spin centres with respect to each other. If, in addition to the dipolar through-space coupling, a through-bond exchange coupling mechanism contributes to the overall coupling both mechanisms can be separated and quantified. Over the last 10 years PELDOR has emerged as a powerful new biophysical method without size restriction to the biomolecule to be studied, and has been applied to a large variety of nucleic acids as well as proteins and protein complexes in solution or within membranes. Small nitroxide spin labels, paramagnetic metal ions, amino acid radicals or intrinsic clusters and cofactor radicals have been used as spin centres.

Orthogonal spin labeling using click chemistry for in vitro and in vivo applications

2017 ◽  
Vol 275 ◽  
pp. 38-45 ◽  
Author(s):  
Svetlana Kucher ◽  
Sergei Korneev ◽  
Swati Tyagi ◽  
Ronja Apfelbaum ◽  
Dina Grohmann ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Spin Labeling

Nitroxide-labeled pyrimidines for non-covalent spin-labeling of abasic sites in DNA and RNA duplexes

2014 ◽  
Vol 12 (37) ◽  
pp. 7366-7374 ◽  
Author(s):  
Sandip A. Shelke ◽  
Gunnar B. Sandholt ◽  
Snorri Th. Sigurdsson
Keyword(s):  
Spin Labeling ◽  
Spin Labels ◽  
Abasic Site ◽  
Abasic Sites ◽  
Dna And Rna ◽  
Rna Duplexes ◽  
Nitroxide Spin Labels ◽  
Uracil Derivative

Of ten new pyrimidine-derived nitroxide spin labels, an N1-ethylamino triazole-linked uracil derivative binds fully to both DNA and RNA duplexes containing an abasic site, as determined by CW-EPR.

Conformational changes of the chaperone SecB upon binding to a model substrate – bovine pancreatic trypsin inhibitor (BPTI)

2011 ◽  
Vol 392 (10) ◽  
pp. 849-858 ◽  
Author(s):  
Michaela M. Haimann ◽  
Yasar Akdogan ◽  
Reinhard Philipp ◽  
Raghavan Varadarajan ◽  
Dariush Hinderberger ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Trypsin Inhibitor ◽  
Tertiary Structure ◽  
Substrate Binding ◽  
Spin Labeling ◽  
Bovine Pancreatic Trypsin Inhibitor ◽  
Spin Labels ◽  
Distance Measurements ◽  
Pancreatic Trypsin Inhibitor

Abstract SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy

2008 ◽  
Vol 52 (1) ◽  
pp. 147-148 ◽  
Author(s):  
M. Flaender ◽  
G. Sicoli ◽  
Th. Fontecave ◽  
G. Mathis ◽  
C. Saint-Pierre ◽  
...  
Keyword(s):  
Click Chemistry ◽  
Dna Probes ◽  
Spin Labels ◽  
Site Specific ◽  
Nitroxide Spin Labels

scholarly journals Reconciling Membrane Protein Simulations with Experimental DEER Spectroscopy Data

2020 ◽  
Author(s):  
Shriyaa Mittal ◽  
Diwakar Shukla
Keyword(s):  
Membrane Proteins ◽  
Conformational Dynamics ◽  
Md Simulations ◽  
Covalent Modification ◽  
Mitigation Strategies ◽  
Residue Pair ◽  
Spin Labels ◽  
Distance Distributions ◽  
Nitroxide Spin Labels ◽  
Double Electron Electron Resonance

AbstractSpectroscopy experiments are crucial to study membrane proteins for which traditional structure determination methods still prove challenging. Double electron-electron resonance (DEER) spectroscopy experiments provide protein residue-pair distance distributions that are indicative of their conformational heterogeneity. Atomistic molecular dynamics (MD) simulations are another tool that have proved vital to study the structural dynamics of membrane proteins such as to identify inward-open, occluded, and outward-open conformations of transporter membrane proteins, among other partially open or closed states of the protein. Yet, studies have reported that there is no direct consensus between distributional data from DEER experiments and MD simulations, which has challenged validation of structures obtained from long-timescale simulations and using simulations to design experiments. Current coping strategies for comparisons rely on heuristics, such as mapping nearest matching peaks between two ensembles or biased simulations. Here we examine the differences in residue-pair distance distributions arising due to choice of membrane around the protein and covalent modification of a pair of residues to nitroxide spin labels in DEER experiments. Through comparing MD simulations of two proteins, PepTSo and LeuT - both of which have been characterized using DEER experiments previously - we show that the proteins’ dynamics are similar despite the choice of the detergent micelle as a membrane mimetic in DEER experiments. On the other hand, covalently modified residues show slight local differences in their dynamics and a huge divergence when the spin labels’ anointed oxygen atom pair distances are measured rather than protein backbone distances. Given the computational expense associated with pairwise MTSSL labeled MD simulations, we examine the use of biased simulations to explore the conformational dynamics of the spin labels only to reveal that such simulations alter the underlying protein dynamics. Our study identifies the main cause for the mismatch between DEER experiments and MD simulations and will accelerate developing potential mitigation strategies to improve simulation observables match with DEER spectroscopy experiments.

Comparison of Ionic and Non-Ionic Nitroxide Spin Labels for Urographic Enhancement in Magnetic Resonance Imaging

1987 ◽  
Vol 28 (5) ◽  
pp. 593-600 ◽  
Author(s):  
W. Grodd ◽  
H. Paajanen ◽  
U. G. Eriksson ◽  
D. Revel ◽  
F. Terrier ◽  
...  
Keyword(s):  
Water Solubility ◽  
Spin Echo ◽  
Renal Medulla ◽  
Acute Tolerance ◽  
Spin Labels ◽  
Resonance Imaging ◽  
Enhancing Effect ◽  
Nitroxide Spin Labels ◽  
Good Contrast

Two nitroxide spin labels (NSL) were compared for in vitro relaxivity and in normal rats for efficiency of urographic enhancement. One of the NSL, PCA, a pyrrolidinyl agent, was ionic and one, NAT, was a non-ionic pyrrolidinyl NSL with multiple hydroxyl substituents for water solubility. Using both NSLs the renal medulla and papilla were noted to show greater contrast enhancement than the cortex, with a maximum enhancing effect between 5 and 15 minutes. Using doses of 1.0 and 2.5 mmol/kg, more than 100 per cent increases in spin echo intensities above the baseline were observed. The lowest tested dose of 0.1 mmol/kg showed an easily detectable enhancing effect for NAT. The good contrast enhancing properties of NAT, considered together with its better acute tolerance, justifies further investigation of this non-ionic compound.

Determination of in vitro „anti-aging„-effects of Ganoderma pfeifferi

Planta Medica ◽  
2010 ◽  
Vol 76 (12) ◽  
Author(s):  
W Jülich ◽  
J Pörksen ◽  
H Welzel ◽  
U Lindequist
Keyword(s):  
Aging Effects
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