scholarly journals A novel strategy for site selective spin-labeling to investigate bioactive entities by DNP and EPR spectroscopy

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin Herr ◽  
Max Fleckenstein ◽  
Martin Brodrecht ◽  
Mark V. Höfler ◽  
Henrike Heise ◽  
...  
Keyword(s):  
Solid State ◽  
Epr Spectroscopy ◽  
Spin Label ◽  
Tertiary Structure ◽  
Spin Labeling ◽  
Disulfide Bridge ◽  
Bioactive Molecules ◽  
Spin Labels ◽  
Time Saving ◽  
Site Selective

AbstractA novel specific spin-labeling strategy for bioactive molecules is presented for eptifibatide (integrilin) an antiplatelet aggregation inhibitor, which derives from the venom of certain rattlesnakes. By specifically labeling the disulfide bridge this molecule becomes accessible for analytical techniques such as Electron Paramagnetic Resonance (EPR) and solid state Dynamic Nuclear Polarization (DNP). The necessary spin-label was synthesized and inserted into the disulfide bridge of eptifibatide via reductive followed by insertion by a double Michael addition under physiological conditions. This procedure is universally applicable for disulfide containing biomolecules and is expected to preserve their tertiary structure with minimal change due to the small size of the label and restoring of the previous disulfide connection. HPLC and MS analysis show the successful introduction of the spin label and EPR spectroscopy confirms its activity. DNP-enhanced solid state NMR experiments show signal enhancement factors of up to 19 in 13C CP MAS experiments which corresponds to time saving factors of up to 361. This clearly shows the high potential of our new spin labeling strategy for the introduction of site selective radical spin labels into biomolecules and biosolids without compromising its conformational integrity for structural investigations employing solid-state DNP or advanced EPR techniques.

Some Applications of Spin Labels in the Study of Serum Albumin and Biomembranes

2008 ◽  
Vol 59 (11) ◽  
Author(s):  
Gheorghe Benga ◽  
Ioana-Costina Dansoreanu ◽  
Maria Frangopol ◽  
Petre T. Frangopol
Keyword(s):  
Serum Albumin ◽  
Spin Label ◽  
Human Liver ◽  
Liver Microsomes ◽  
Egg Yolk ◽  
Spin Labeling ◽  
Spin Labels ◽  
Sh Groups ◽  
Low Concentrations ◽  
Computerized System

The main contributions of the groups lead by the two main authors (Gh.B and P.T.F.) in the field of spin labeling ESR in studies of serum albumin and of some biomembranes are briefly described, mentioning some priorities. These are: the description in Romanian publications of the methodology for using spin labels in studies of proteins and biomembranes using the type ART-6 ESR spectrometer manufactured in Romania; the adaptation to this ESR spectrometer of a computerized system for the acqusition and processing of data; the application of a newly synthesized spin label, reactive with SH groups, in the study of serum albumin; the finding of the effects of 25-hydroxycholesterol at low concentrations (0,5-10 %) on egg yolk lecitin; the finding of a relatively high fluidity of human liver microsomes.

Nitroxides and nucleic acids: Chemistry and electron paramagnetic resonance (EPR) spectroscopy

2011 ◽  
Vol 83 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Snorri Th. Sigurdsson
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Nucleic Acids ◽  
Epr Spectroscopy ◽  
Noncovalent Interactions ◽  
Spin Labeling ◽  
Structural Features ◽  
Spin Labels ◽  
Abasic Site ◽  
Paramagnetic Resonance ◽  
Electron Paramagnetic

Electron paramagnetic resonance (EPR) spectroscopy has increasingly been applied for the study of nucleic acid structure and dynamics. Such studies require incorporation of free radicals (spin labels) into the biopolymer. The labels can be incorporated during chemical synthesis of the oligomer (phosphoramidite approach) or postsynthetically, by reaction of a spin-labeling reagent with a reactive functional group on the oligonucleotide. Incorporation of the rigid nitroxide spin label Ç is an example of the phosphoramidite method, and reaction of a spin-labeled azide with an alkyne-modified oligomer to yield a triazole-derived, spin-labeled nucleotide illustrates the postsynthetic spin-labeling strategy. Characterization and application of these labels to study structural features of DNA by EPR spectroscopy is discussed. Finally, a new spin-labeling strategy is described for nucleic acids that relies on noncovalent interactions between a spin-labeled nucleobase and an abasic site in duplex DNA.

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.

scholarly journals Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy

Molecules ◽  
2019 ◽  
Vol 24 (15) ◽  
pp. 2735 ◽  
Author(s):  
J. Jacques Jassoy ◽  
Caspar A. Heubach ◽  
Tobias Hett ◽  
Frédéric Bernhard ◽  
Florian R. Haege ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Spin Labeling ◽  
Single Frequency ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Site Directed Spin Labeling ◽  
Site Selective ◽  
Electron Paramagnetic

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

scholarly journals The Molten Globule State of Maltose-Binding Protein: Structural and Thermodynamic Characterization by EPR Spectroscopy and Isothermal Titration Calorimetry

2020 ◽  
Vol 51 (9-10) ◽  
pp. 877-886
Author(s):  
Chen Nickolaus ◽  
Carolyn Vargas ◽  
Jörg Reichenwallner ◽  
Mohammed Chakour ◽  
Benjamin Selmke ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Tertiary Structure ◽  
Binding Protein ◽  
Molten Globule ◽  
Spin Labeling ◽  
Maltose Binding Protein ◽  
Titration Calorimetry ◽  
Native State ◽  
Paramagnetic Resonance ◽  
Distance Information

Abstract Employing site-directed spin labeling (SDSL), the structure of maltose-binding protein (MBP) had previously been studied in the native state by electron paramagnetic resonance (EPR) spectroscopy. Several spin-labeled double cysteine mutants were distributed all over the structure of this cysteine-free protein and revealed distance information between the nitroxide residues from double electron–electron resonance (DEER). The results were in good agreement with the known X-ray structure. We have now extended these studies to the molten globule (MG) state, a folding intermediate, which can be stabilized around pH 3 and that is characterized by secondary but hardly any tertiary structure. Instead of clearly defined distance features as found in the native state, several additional characteristics indicate that the MG structure of MBP contains different polypeptide chain and domain orientations. MBP is also known to bind its substrate maltose even in MG state although with lower affinity. Additionally, we have now created new mutants allowing for spin labeling at or near the active site. Our data confirm an already preformed ligand site structure in the MG explaining its substrate binding capability and thus most probably serving as a nucleation center for the final native structure.

A semi-rigid isoindoline-derived nitroxide spin label for RNA

2018 ◽  
Vol 16 (5) ◽  
pp. 816-824 ◽  
Author(s):  
Dnyaneshwar B. Gophane ◽  
Burkhard Endeward ◽  
Thomas F. Prisner ◽  
Snorri Th. Sigurdsson
Keyword(s):  
Epr Spectroscopy ◽  
Spin Label ◽  
Spin Labels ◽  
Pulsed Epr ◽  
Rna Duplexes ◽  
Limited Motion

The new semi-rigid spin label ImUm showed limited motion in RNA duplexes and accurate distances between two spin labels in RNA duplexes were obtained by pulsed EPR spectroscopy.

scholarly journals Organophotocatalytic selective deuterodehalogenation of aryl or alkyl chlorides

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yanjun Li ◽  
Ziqi Ye ◽  
Yu-Mei Lin ◽  
Yan Liu ◽  
Yumeng Zhang ◽  
...  
Keyword(s):  
Room Temperature ◽  
Sodium Formate ◽  
Bioactive Molecules ◽  
Co Catalyst ◽  
Aryl Chlorides ◽  
Aryl Amine ◽  
Drug Molecules ◽  
Photocatalytic System ◽  
Site Selective ◽  
Environmentally Friendly Method

AbstractDevelopment of practical deuteration reactions is highly valuable for organic synthesis, analytic chemistry and pharmaceutic chemistry. Deuterodehalogenation of organic chlorides tends to be an attractive strategy but remains a challenging task. We here develop a photocatalytic system consisting of an aryl-amine photocatalyst and a disulfide co-catalyst in the presence of sodium formate as an electron and hydrogen donor. Accordingly, many aryl chlorides, alkyl chlorides, and other halides are converted to deuterated products at room temperature in air (>90 examples, up to 99% D-incorporation). The mechanistic studies reveal that the aryl amine serves as reducing photoredox catalyst to initiate cleavage of the C-Cl bond, at the same time as energy transfer catalyst to induce homolysis of the disulfide for consequent deuterium transfer process. This economic and environmentally-friendly method can be used for site-selective D-labeling of a number of bioactive molecules and direct H/D exchange of some drug molecules.

Spin Label Study of Apomembranes and Purple Membranes

1993 ◽  
Vol 48 (5-6) ◽  
pp. 500-503
Author(s):  
Tzvetana R. Lazarova ◽  
Maya Y. Velitchkova
Keyword(s):  
Fatty Acids ◽  
Lipid Bilayer ◽  
Spin Label ◽  
Hyperfine Splitting ◽  
Esr Spectra ◽  
Spin Labels ◽  
Label Study ◽  
Splitting Parameter ◽  
Induced Changes ◽  
Purple Membranes

Abstract Three spin-labelled fatty acids were used to detect the dynamics of lipid bilayer of apomem branes and purple membranes. It was found that ESR spectra of spin labels bound to apo­ membranes showed a temperature-induced changes rather similar to those seen with purple membranes. At the same time, however, the values of hyperfine splitting parameter 2Tm were lower as compared to purple membranes. The results pointed out that the removal of the retinal from purple membranes affects the dynamics of lipid bilayer and apomembranes were more rigid structure than those of purple membranes.

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