scholarly journals DNA complexes with human apurinic/apyrimidinic endonuclease 1: structural insights revealed by pulsed dipolar EPR with orthogonal spin labeling

2019 ◽  
Vol 47 (15) ◽  
pp. 7767-7780 ◽  
Author(s):  
Olesya A Krumkacheva ◽  
Georgiy Yu Shevelev ◽  
Alexander A Lomzov ◽  
Nadezhda S Dyrkheeva ◽  
Andrey A Kuzhelev ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Md Simulations ◽  
Dna Lesions ◽  
Spin Labels ◽  
Paramagnetic Resonance ◽  
Applied Pulse ◽  
Oligonucleotide Duplex ◽  
Nitroxide Spin Labels ◽  
Ap Sites ◽  
Electron Paramagnetic

Abstract A DNA molecule is under continuous influence of endogenous and exogenous damaging factors, which produce a variety of DNA lesions. Apurinic/apyrimidinic sites (abasic or AP sites) are among the most common DNA lesions. In this work, we applied pulse dipolar electron paramagnetic resonance (EPR) spectroscopy in combination with molecular dynamics (MD) simulations to investigate in-depth conformational changes in DNA containing an AP site and in a complex of this DNA with AP endonuclease 1 (APE1). For this purpose, triarylmethyl (TAM)-based spin labels were attached to the 5′ ends of an oligonucleotide duplex, and nitroxide spin labels were introduced into APE1. In this way, we created a system that enabled monitoring the conformational changes of the main APE1 substrate by EPR. In addition, we were able to trace substrate-to-product transformation in this system. The use of different (orthogonal) spin labels in the enzyme and in the DNA substrate has a crucial advantage allowing for detailed investigation of local damage and conformational changes in AP-DNA alone and in its complex with APE1.

scholarly journals The Internal Dynamics of Mini c TAR DNA Probed by Electron Paramagnetic Resonance of Nitroxide Spin-Labels at the Lower Stem, the Loop, and the Bulge

Biochemistry ◽  
2012 ◽  
Vol 51 (43) ◽  
pp. 8530-8541 ◽  
Author(s):  
Yan Sun ◽  
Ziwei Zhang ◽  
Vladimir M. Grigoryants ◽  
William K. Myers ◽  
Fei Liu ◽  
...  
Keyword(s):  
Electron Paramagnetic Resonance ◽  
Spin Labels ◽  
Internal Dynamics ◽  
Paramagnetic Resonance ◽  
Nitroxide Spin Labels ◽  
Electron Paramagnetic ◽  
Lower Stem

scholarly journals Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels

2021 ◽  
Author(s):  
Igor Tkach ◽  
Ulf Diederichsen ◽  
Marina Bennati
Keyword(s):  
Molecular Level ◽  
Dipolar Interaction ◽  
Spin Labels ◽  
Paramagnetic Centers ◽  
Structural Adaptation ◽  
Paramagnetic Resonance ◽  
Transmembrane Peptides ◽  
Distance Distributions ◽  
Lipid Environment ◽  
Electron Paramagnetic

AbstractElectron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5–10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes. Graphical Abstract

scholarly journals Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution

2020 ◽  
Vol 48 (9) ◽  
pp. e49-e49 ◽  
Author(s):  
Shreya Ghosh ◽  
Matthew J Lawless ◽  
Hanna J Brubaker ◽  
Kevin Singewald ◽  
Michael R Kurpiewski ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Conformational Changes ◽  
Continuous Wave ◽  
Md Simulations ◽  
Distance Measurements ◽  
Paramagnetic Resonance ◽  
Pulsed Epr ◽  
Distance Distributions ◽  
Dna Backbone ◽  
Double Electron Electron Resonance

Abstract Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2′dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

scholarly journals Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

2020 ◽  
Vol 117 (33) ◽  
pp. 19720-19730 ◽  
Author(s):  
Yifan Wang ◽  
Kathy Fange Liu ◽  
Yu Yang ◽  
Ian Davis ◽  
Aimin Liu
Keyword(s):  
Conformational Changes ◽  
Quinolinic Acid ◽  
De Novo ◽  
Crystalline Lens ◽  
Paramagnetic Resonance ◽  
Time Resolved ◽  
X Ray Crystallography ◽  
Protein Conformational Changes ◽  
Catalytic Intermediates ◽  
Electron Paramagnetic

The synthesis of quinolinic acid from tryptophan is a critical step in the de novo biosynthesis of nicotinamide adenine dinucleotide (NAD+) in mammals. Herein, the nonheme iron-based 3-hydroxyanthranilate-3,4-dioxygenase responsible for quinolinic acid production was studied by performing time-resolvedin crystalloreactions monitored by UV-vis microspectroscopy, electron paramagnetic resonance (EPR) spectroscopy, and X-ray crystallography. Seven catalytic intermediates were kinetically and structurally resolved in the crystalline state, and each accompanies protein conformational changes at the active site. Among them, a monooxygenated, seven-membered lactone intermediate as a monodentate ligand of the iron center at 1.59-Å resolution was captured, which presumably corresponds to a substrate-based radical species observed by EPR using a slurry of small-sized single crystals. Other structural snapshots determined at around 2.0-Å resolution include monodentate and subsequently bidentate coordinated substrate, superoxo, alkylperoxo, and two metal-bound enol tautomers of the unstable dioxygenase product. These results reveal a detailed stepwise O-atom transfer dioxygenase mechanism along with potential isomerization activity that fine-tunes product profiling and affects the production of quinolinic acid at a junction of the metabolic pathway.

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