Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Song, Likai; Liu, Zhanglong; Kaur, Pavanjeet; Esquiaqui, Jackie M.; Hunter, Robert I.; Hill, Stephen; Smith, Graham; Fanucci, Gail E.

doi:10.1016/j.jmr.2016.02.007

Cited by 25 publications

(36 citation statements)

References 74 publications

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“…To overcome this problem, a cavity-less W-band EPR system in which more sample volume ($10 ll) can be mounted has been reported to demonstrate the concentration sensitivity of 2 lM for TEMPO radical. 38 On the other hand, the concentration sensitivity achieved in this study was $1.4 mM for aquometmyoglobin, worse than the above-mentioned value and those needed for other spectroscopic methods such as solution NMR and UV-Vis absorption. However, the EPR spectra of aquomet-myoglobin was so broad (>2.5 T) that we would expect better concentration sensitivity of less than 1 lM for EPR species exhibiting narrower line width such as radicals.…”

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confidence: 64%

Force detection of high-frequency electron paramagnetic resonance spectroscopy of microliter solution sample

Okamoto

Takahashi

Ohmichi

et al. 2018

Applied Physics Letters

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Force detection of magnetic resonance is now able to attain extremely high spin sensitivity. In these setups, microcantilevers were usually used as a sensitive force sensor and, in most cases, have been applied to solid-state samples such as paramagnetic impurities in solids. On the other hand, there are now growing demands for their applications to liquid-state samples in the research areas of life science because many proteins and enzymes are biofunctionally active only in solutions, where they interact with the surrounding water molecules. In this letter, we present an electron paramagnetic resonance (EPR) technique for solution samples using a SiNx nanomembrane and report high-frequency EPR spectroscopy of a microliter-volume frozen solution sample of hemin and myoglobin at multiple frequencies up to 350 GHz. This technique would be particularly useful to obtain more detailed insight into the electronic structure of metalloproteins/metalloenzymes under biologically active conditions.

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confidence: 64%

Force detection of high-frequency electron paramagnetic resonance spectroscopy of microliter solution sample

Okamoto

Takahashi

Ohmichi

et al. 2018

Applied Physics Letters

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“…To examine the lateral ordering of lipids, EPR spectra were recorded on a W-band 94 GHz continuous-wave and pulsed EPR spectrometer (52), with a quasi-optical transmission design, at the NHMFL. The EPR measurements were performed using a nonresonant sample holder containing a thin-cylindrical layer of sample of 20-mm diameter, 0.17-mm thickness, and 53-mL volume (53). The sample holder is operating in induction mode, i.e., samples are irradiated with linearly polarized microwaves and signals are detected as the orthogonal mode of the circularly polarized radiation that is emitted from the sample.…”

Section: Lipid Lateral Ordering Measurementsmentioning

confidence: 99%

Selective Membrane Disruption Mechanism of an Antibacterial γ-AApeptide Defined by EPR Spectroscopy

Kaur

Cai

et al. 2016

Biophysical Journal

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γ-AApeptides are a new class of antibacterial peptidomimetics that are not prone to antibiotic resistance and are highly resistant to protease degradation. It is not clear how γ-AApeptides interact with bacterial membranes and alter lipid assembly, but such information is essential to understanding their antimicrobial activities and guiding future design of more potent and specific antimicrobial agents. Using electron paramagnetic resonance techniques, we characterized the membrane interaction and destabilizing mechanism of a lipo-cyclic-γ-AApeptide (AA1), which has broad-spectrum antibacterial activities. The analyses revealed that AA1 binding increases the membrane permeability of POPC/POPG liposomes, which mimic negatively charged bacterial membranes. AA1 binding also inhibits membrane fluidity and reduces solvent accessibility around the lipid headgroup region. Moreover, AA1 interacts strongly with POPC/POPG liposomes, inducing significant lipid lateral-ordering and membrane thinning. In contrast, minimal membrane property changes were observed upon AA1 binding for liposomes mimicking mammalian cell membranes, which consist of neutral lipids and cholesterol. Our findings suggest that AA1 interacts and disrupts bacterial membranes through a carpet-like mechanism. The results showed that the intrinsic features of γ-AApeptides are important for their ability to disrupt bacterial membranes selectively, the implications of which extend to developing new antibacterial biomaterials.

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“…-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 modern EPR spectroscopy to the mm-and sub-mm wavelength and 15 T Zeeman field regions. Sub-micromolar concentrations of paramagnetic molecules such as nitroxide spin-labeled protein complexes have become sufficient to characterize reaction intermediates-offering new application possibilities in biochemistry and molecular biology [17,28,29]. Moreover, for multifrequency EPR experiments on frozen solutions typical sample volumes are 250 µL (S-band), 150 µL (X-band), 10 µL (Q-band) and 1 µL (W-band), see [649][650][651][652][653].…”

Section: Discussionmentioning

confidence: 99%

“…This principal sensitivity advantage of EPR is even more accentuated when going from standard X-band EPR to high-field EPR operating, for example, in the mm and sub-mm frequency domains. Minute quantities of samples (µM versus mM concentrations [28,29]) have become sufficient to characterize stable and short-lived transient reaction intermediates of complex molecular systems-offering very interesting applications for biochemists and molecular biologists [1].…”

Section: Nmr Versus Epr Spectroscopy At High Magnetic Fieldsmentioning

confidence: 99%

“…In terms of instrumental development beyond currently available commercial high-field EPR spectrometers (see, www.bruker.com) the research group of G. Smith at the University of St Andrews conducts an ambitious project called "HIPER" (funded under the UK Basic Technology program). The group has designed a unique high-power quasioptical W-band (94 GHz, 1 kW) pulse EPR machine [28,29] featuring π/2 pulses as short as 5 ns and a detection bandwidth of 1 GHz in nonresonant sample holders operating in induction mode and at low temperatures. Low-power microwave pulses can be as short as 200 ps, and kilowatt pulses as short as 1.5 ns with timing resolution of a few hundred ps.…”

Section: High-field Epr Instrumentationmentioning

confidence: 99%

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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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Toward increased concentration sensitivity for continuous wave EPR investigations of spin-labeled biological macromolecules at high fields

Cited by 25 publications

References 74 publications

Force detection of high-frequency electron paramagnetic resonance spectroscopy of microliter solution sample

Force detection of high-frequency electron paramagnetic resonance spectroscopy of microliter solution sample

Selective Membrane Disruption Mechanism of an Antibacterial γ-AApeptide Defined by EPR Spectroscopy

Biomolecular EPR Meets NMR at High Magnetic Fields

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