EPR Spectroscopy of Nitroxide Spin Probes

Bordignon, Enrica

doi:10.1002/9780470034590.emrstm1513

Cited by 52 publications

(51 citation statements)

References 86 publications

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“…SDSL-EPR spectroscopy is a non-destructive technique that provides details on protein structure and flexibility over a wide-range of temperatures and timescales [35][36][37][38]. Proteins can be studied in their native environment, that is in membranes, in cellular extract and also inside cells [39].…”

Section: Cys Variants Were Generated To Selectively Label Distinct Rementioning

confidence: 99%

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

et al. 2020

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UreG is a P-loop GTP hydrolase involved in the maturation of nickel-containing urease, an essential enzyme found in plants, fungi, bacteria, and archaea. This protein couples the hydrolysis of GTP to the delivery of Ni(II) into the active site of apo-urease, interacting with other urease chaperones in a multi-protein complex necessary for enzyme activation. Whereas the conformation of Helicobacter pylori (Hp) UreG was solved by crystallography when it is in complex with two other chaperones, in solution the protein was found in a disordered and flexible form, defining it as an intrinsically disordered enzyme and indicating that the well-folded structure found in the crystal state does not fully reflect the behavior of the protein in solution. Here, isothermal titration calorimetry and site-directed spin labeling coupled to electron paramagnetic spectroscopy were successfully combined to investigate HpUreG structural dynamics in solution and the effect of Ni(II) and GTP on protein mobility. The results demonstrate that, although the protein maintains a flexible behavior in the metal and nucleotide bound forms, concomitant addition of Ni(II) and GTP exerts a structural change through the crosstalk of different protein regions.

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Section: Cys Variants Were Generated To Selectively Label Distinct Rementioning

confidence: 99%

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

et al. 2020

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“…However, it requires the introduction of paramagnetic spin centres in the protein of interest, for example by site‐specifically attaching spin labels to cysteines introduced at desired sites, with specificity requiring the replacement of all natural cysteines. The most common spin label is MTSL (1‐Oxyl‐2,2,5,5‐tetramethyl‐∆3‐pyrroline‐3‐methyl) (methanethiosulfonate), a nitroxide label with spin 1/2 [26]. For specific applications, maleimide‐functionalized chelated Gd 3+ spin labels (spin 7/2) [27] were also used in the ABC transporter field, as described in the following.…”

Section: Electron Paramagnetic Resonance Spectroscopymentioning

confidence: 99%

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

et al. 2020

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ATP-binding cassette (ABC) exporters have been studied now for more than four decades, and recent structural investigation has produced a large number of protein database entries. Yet, important questions about how ABC exporters function at the molecular level remain debated, such as which are the molecular recognition hotspots and the allosteric couplings dynamically regulating the communication between the catalytic cycle and the export of substrates. This conundrum mainly arises from technical limitations confining all research to in vitro analysis of ABC transporters in detergent solutions or embedded in membrane-mimicking environments. Therefore, a largely unanswered question is how ABC exporters operate in situ, namely in the native membrane context of a metabolically active cell. This review focuses on novel mechanistic insights into type I ABC exporters gained through a unique combination of structure determination, biochemical characterization, generation of conformation-specific nanobodies/sybodies and double electron-electron resonance.

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“…Aminoxyl radicals (nitroxides) are stable radicals used extensively in EPR spectroscopy as a spin label or local reporter group in solution and solid phase samples, for a recent review see [586]. Nitroxide radicals contain an NMR active 14 N (or 15 N) nucleus whose spectroscopic properties (hyperfine/quadrupole coupling) are dependent on the nitroxide's local environment, see Section 5.3.…”

Section: Nitrogen Ednmr On Nitroxide Radicals In Organic Solventsmentioning

confidence: 99%

Biomolecular EPR Meets NMR at High Magnetic Fields

et al. 2018

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In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth.

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EPR Spectroscopy of Nitroxide Spin Probes

Cited by 52 publications

References 86 publications

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

Nickel and GTP Modulate Helicobacter pylori UreG Structural Flexibility

From in vitro towards in situ: structure‐based investigation of ABC exporters by electron paramagnetic resonance spectroscopy

Biomolecular EPR Meets NMR at High Magnetic Fields

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