scholarly journals A Comparison of Cysteine-Conjugated Nitroxide Spin Labels for Pulse Dipolar EPR Spectroscopy

Molecules ◽  
2021 ◽  
Vol 26 (24) ◽  
pp. 7534
Author(s):  
Katrin Ackermann ◽  
Alexandra Chapman ◽  
Bela E. Bode
Keyword(s):  
Materials Science ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Protein Structures ◽  
Nitroxide Radical ◽  
Structural Heterogeneity ◽  
Site Directed Mutagenesis ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Side Chains ◽  
Distance Distributions

The structure-function and materials paradigms drive research on the understanding of structures and structural heterogeneity of molecules and solids from materials science to structural biology. Functional insights into complex architectures are often gained from a suite of complementary physicochemical methods. In the context of biomacromolecular structures, the use of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) has become increasingly popular. The main interest in PDS is providing long-range nanometre distance distributions that allow for identifying macromolecular topologies, validating structural models and conformational transitions as well as docking of quaternary complexes. Most commonly, cysteines are introduced into protein structures by site-directed mutagenesis and modified site-specifically to a spin-labelled side-chain such as a stable nitroxide radical. In this contribution, we investigate labelling by four different commercial labelling agents that react through different sulfur-specific reactions. Further, the distance distributions obtained are between spin-bearing moieties and need to be related to the protein structure via modelling approaches. Here, we compare two different approaches to modelling these distributions for all four side-chains. The results indicate that there are significant differences in the optimum labelling procedure. All four spin-labels show differences in the ease of labelling and purification. Further challenges arise from the different tether lengths and rotamers of spin-labelled side-chains; both influence the modelling and translation into structures. Our comparison indicates that the spin-label with the shortest tether in the spin-labelled side-group, (bis-(2,2,5,5-Tetramethyl-3-imidazoline-1-oxyl-4-yl) disulfide, may be underappreciated and could increase the resolution of structural studies by PDS if labelling conditions are optimised accordingly.

scholarly journals Protein fold prediction using simulated DEER distance distributions and decay traces

2019 ◽  
Author(s):  
Diego del Alamo ◽  
Maxx Tessmer ◽  
Richard Stein ◽  
Jimmy B. Feix ◽  
Hassane S. Mchaourab ◽  
...  
Keyword(s):  
Experimental Data ◽  
Structure Prediction ◽  
De Novo ◽  
Time Window ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Coarse Grained ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Software Suite ◽  
Distance Distributions

AbstractDespite advances in sampling and scoring strategies, Monte Carlo modeling methods still struggle to accurately predict de novo the structures of large proteins, membrane proteins, or proteins of complex topologies. Previous approaches have addressed these shortcomings by leveraging sparse distance data gathered using site-directed spin labeling and electron paramagnetic resonance spectroscopy (SDSL-EPR) to improve protein structure prediction and refinement outcomes. However, existing computational implementations must choose between coarse-grained models of the spin label that lower the resolution and explicit models that lead to resource-intense simulations. Existing methods are further limited by their reliance on distance distributions, which are calculated from a primary refocused echo decay signal and may contain artifacts introduced during this processing step. Here, we addressed these challenges by developing RosettaDEER, a scoring method within the Rosetta software suite capable of simulating distance distributions and echo decay traces between spin labels fast enough to fold proteins de novo. We demonstrate that the accuracy of resulting distance distributions match or exceed those generated by more computationally intensive methods. Moreover, decay traces generated from these distributions recapitulate intermolecular background coupling parameters, allowing RosettaDEER to discriminate between poorly-folded and native-like models even when the time window of EPR data collection is truncated, rendering them unsuitable for accurate transformation into distance distributions. Finally, we demonstrate that one decay trace per nine residues is sufficient to predict the folds of Bax and the C-terminus of ExoU, two soluble proteins with surface-exposed amphipathic structural features that prevent the Rosetta energy function from correctly identifying native-like models in the absence of experimental data. These benchmarking results confirm that RosettaDEER can effectively leverage sparse experimental data for a wide array of modeling applications built into the Rosetta software suite.

scholarly journals Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion

2017 ◽  
Vol 292 (18) ◽  
pp. 7598-7606 ◽  
Author(s):  
Sylwia Kacprzak ◽  
Ibrahim Njimona ◽  
Anja Renz ◽  
Juan Feng ◽  
Edward Reijerse ◽  
...  
Keyword(s):  
Histidine Kinase ◽  
Double Resonance ◽  
Red Light ◽  
Full Length ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Pulsed Epr ◽  
Protein Dimer ◽  
Distance Distributions ◽  
Structural Details

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

scholarly journals Design of an Antioxidant Nitrone Scaffold Based on Plant Pigments

2021 ◽  
Author(s):  
Amanda Capistrano Pinheiro ◽  
Rodrigo Boni Fazzi ◽  
Larissa Cerrato Esteves ◽  
Caroline Oliveira Machado ◽  
Felipe Augusto Dörr ◽  
...  
Keyword(s):  
Transient Absorption ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Hydrolytic Stability ◽  
Nitroxide Radical ◽  
Natural Antioxidants ◽  
Transient Absorption Spectroscopy ◽  
Alkaline Media ◽  
Phenolic Antioxidants ◽  
Radical Scavenger ◽  
Resonance Spectroscopy

Nitrones derived from natural antioxidants are emerging as highly specific therapeutics for the treatment of various human diseases, including stroke, neurodegenerative pathologies, and cancer. However, the development of useful pseudo-natural nitrones requires the judicious choice of a secondary metabolite as precursor. Betalains are nitrogen-containing natural pigments that exhibit marked antioxidant and pharmacological properties and, hence, are ideal candidates for the design of multifunctional nitrones. In this work, we describe the semisynthesis and properties of a biocompatible and antioxidant betalain-nitrone named OxiBeet. This bio-based compound is a better radical scavenger than ascorbic acid, gallic acid and most non-phenolic antioxidants and undergoes concerted proton-coupled electron transfer. The autoxidation of OxiBeet gives rise to a persistent nitroxide radical, which was studied by electron paramagnetic resonance spectroscopy. Femtosecond transient absorption spectroscopy reveals that excited state formation is not required for the oxidation of OxiBeet. The results are compared with those obtained using betanin, a natural betalain, and pBeet, the imine analogue of OxiBeet. The findings in this study will enable the development of antioxidant nitrones based on the novel <i>N</i>-oxide 1,7-diazaheptamethinium scaffold and betalain dyes with enhanced hydrolytic stability in aqueous alkaline media.

scholarly journals Protein functional dynamics from the rigorous global analysis of DEER data: Conditions, components, and conformations

2021 ◽  
Vol 153 (11) ◽  
Author(s):  
Eric J. Hustedt ◽  
Richard A. Stein ◽  
Hassane S. Mchaourab
Keyword(s):  
Spectroscopic Data ◽  
Dipolar Coupling ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Global Analysis ◽  
Experimental Studies ◽  
Spin Labeling ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Functional Dynamics

The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

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.

A radical approach to radicals

2022 ◽  
Vol 78 (1) ◽  
Author(s):  
Youjia Liu ◽  
Malgorzata Biczysko ◽  
Nigel W. Moriarty
Keyword(s):  
Protein Structure ◽  
Structural Information ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Molecular Model ◽  
Structural Parameters ◽  
Spin Labels ◽  
Resonance Spectroscopy ◽  
Paramagnetic Resonance ◽  
Widespread Application ◽  
Electron Paramagnetic

Nitroxide radicals are characterized by a long-lived spin-unpaired electronic ground state and are strongly sensitive to their chemical surroundings. Combined with electron paramagnetic resonance spectroscopy, these electronic features have led to the widespread application of nitroxide derivatives as spin labels for use in studying protein structure and dynamics. Site-directed spin labelling requires the incorporation of nitroxides into the protein structure, leading to a new protein–ligand molecular model. However, in protein crystallographic refinement nitroxides are highly unusual molecules with an atypical chemical composition. Because macromolecular crystallography is almost entirely agnostic to chemical radicals, their structural information is generally less accurate or even erroneous. In this work, proteins that contain an example of a radical compound (Chemical Component Dictionary ID MTN) from the nitroxide family were re-refined by defining its ideal structural parameters based on quantum-chemical calculations. The refinement results show that this procedure improves the MTN ligand geometries, while at the same time retaining higher agreement with experimental data.

scholarly journals In-cell destabilization of a homo-dimeric protein complex detected by DEER spectroscopy

2020 ◽  
Author(s):  
Yin Yang ◽  
Shen-Na Chen ◽  
Feng Yang ◽  
Xia-Yan Li ◽  
Akiva Feintuch ◽  
...  
Keyword(s):  
Protein Structures ◽  
Salt Bridge ◽  
Spin Labels ◽  
Excluded Volume ◽  
Excluded Volume Effects ◽  
Distance Distributions ◽  
The Stability ◽  
Double Electron Electron Resonance ◽  
In Cells

AbstractThe complexity of the cellular medium can affect proteins’ properties and therefore in-cell characterization of proteins is essential. We explored the stability and conformation of BIR1, the first baculoviral IAP repeat domain of X-chromosome-linked inhibitor of apoptosis (XIAP), as a model for a homo-dimer protein in human HeLa cells. We employed double electron-electron resonance (DEER) spectroscopy and labeling with redox stable and rigid Gd3+ spin labels at three protein residues, C12 (flexible region), E22C and N28C (part of helical residues 26–31) in the N-terminal region. In contrast to predictions by excluded volume crowding theory, the dimer-monomer dissociation constant KD was markedly higher in cells than in solution and dilute cell lysate. As expected, this increase was recapitulated under conditions of high salt concentrations given that a conserved salt bridge at the dimer interface is critically required for association. Unexpectedly, however, also the addition of a crowding agent such as Ficoll destabilized the dimer, suggesting that Ficoll forms specific interactions with the monomeric protein. Changes in DEER distance distributions were observed for the E22C site, which displayed reduced conformational freedom in cells. Although overall DEER behaviors at E22C and N28C were compatible with a predicted compaction of disordered protein regions by excluded volume effects, we were unable to reproduce E22C properties in artificially crowded solutions. These results highlight the importance of in-cell DEER measurements to appreciate the complexities of cellular in vivo effects on protein structures and functions.

scholarly journals Design of an Antioxidant Nitrone Scaffold Based on Plant Pigments

2021 ◽  
Author(s):  
Amanda Capistrano Pinheiro ◽  
Rodrigo Boni Fazzi ◽  
Larissa Cerrato Esteves ◽  
Caroline Oliveira Machado ◽  
Felipe Augusto Dörr ◽  
...  
Keyword(s):  
Transient Absorption ◽  
Electron Paramagnetic Resonance Spectroscopy ◽  
Hydrolytic Stability ◽  
Nitroxide Radical ◽  
Natural Antioxidants ◽  
Transient Absorption Spectroscopy ◽  
Alkaline Media ◽  
Phenolic Antioxidants ◽  
Radical Scavenger ◽  
Resonance Spectroscopy

<div> <div> <div> <p>Nitrones derived from natural antioxidants are emerging as highly specific therapeutics against various human diseases, including stroke, neurodegenerative pathologies, and cancer. However, the development of useful pseudo-natural nitrones requires the judicious choice of a secondary metabolite as the precursor. Betalains are nitrogen-containing natural pigments that exhibit marked antioxidant capacity and pharmacological properties and, hence, are ideal candidates for designing multifunctional nitrones. In this work, we describe the semisynthesis and properties of a biocompatible, antioxidant betalain-nitrone called OxiBeet. This bio-based compound is a better radical scavenger than ascorbic acid, gallic acid, and most non-phenolic antioxidants and undergoes concerted proton-coupled electron transfer. The autoxidation of OxiBeet produces a persistent nitroxide radical, which, herein, is studied via electron paramagnetic resonance spectroscopy. In addition, femtosecond transient absorption spectroscopy reveals that excited state formation is not required for the oxidation of OxiBeet. The results are compared with those obtained using betanin, a natural betalain, and pBeet, the imine analog of OxiBeet. The findings of this study will enable the development of antioxidant nitrones based on the novel N-oxide 1,7-diazaheptamethinium scaffold and betalain dyes with enhanced hydrolytic stability in aqueous alkaline media. </p> </div> </div> </div>

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