Pulsed Spectroscopy Research Articles

Enabling structural evolution of intrinsically disordered proteins using pulsed dipolar ESR spectroscopy

Enabling structural evolution of intrinsically disordered proteins using pulsed dipolar ESR spectroscopy

Lateral gating mechanism and plasticity of the β-barrel assembly machinery complex in micelles and Escherichia coli.

The β-barrel assembly machinery (BAM) mediates the folding and insertion of the majority of outer membrane proteins (OMPs) in gram-negative bacteria. BAM is a penta-heterooligomeric complex consisting of the central β-barrel BamA and four interacting lipoproteins BamB, C, D, and E. The conformational switching of BamA between inward-open (IO) and lateral-open (LO) conformations is required for substrate recognition and folding. However, the mechanism for the lateral gating or how the structural details observed in vitro correspond with the cellular environment remains elusive. In this study, we addressed these questions by characterizing the conformational heterogeneity of BamAB, BamACDE, and BamABCDE complexes in detergent micelles and/or Escherichia coli using pulsed dipolar electron spin resonance spectroscopy (PDS). We show that the binding of BamB does not induce any visible changes in BamA, and the BamAB complex exists in the IO conformation. The BamCDE complex induces an IO to LO transition through a coordinated movement along the BamA barrel. However, the extracellular loop 6 (L6) is unaffected by the presence of lipoproteins and exhibits large segmental dynamics extending to the exit pore. PDS experiments with the BamABCDE complex in intact E. coli confirmed the dynamic behavior of both the lateral gate and the L6 in the native environment. Our results demonstrate that the BamCDE complex plays a key role in the function by regulating lateral gating in BamA.

Metal-carbon bonding in early lanthanide substituted cyclopentadienyl complexes probed by pulsed EPR spectroscopy.

We examine lanthanide (Ln)-ligand bonding in a family of early Ln3+ complexes [Ln(Cptt)3] (1-Ln, Ln = La, Ce, Nd, Sm; Cptt = C5H3tBu2-1,3) by pulsed electron paramagnetic resonance (EPR) methods, and provide the first characterization of 1-La and 1-Nd by single crystal XRD, multinuclear NMR, IR and UV/Vis/NIR spectroscopy. We measure electron spin T1 and Tm relaxation times of 12 and 0.2 μs (1-Nd), 89 and 1 μs (1-Ce) and 150 and 1.7 μs (1-Sm), respectively, at 5 K: the T1 relaxation of 1-Nd is more than 102 times faster than its valence isoelectronic uranium analogue. 13C and 1H hyperfine sublevel correlation (HYSCORE) spectroscopy reveals that the extent of covalency is negligible in these Ln compounds, with much smaller hyperfine interactions than observed for equivalent actinide (Th and U) complexes. This is corroborated by ab initio calculations, confirming the predominant electrostatic nature of the metal-ligand bonding in these complexes.

Spin labels for 19F ENDOR distance determination: resolution, sensitivity and distance predictability.

19F electron-nuclear double resonance (ENDOR) has emerged as an attractive method for determining distance distributions in biomolecules in the range of 0.7-2 nm, which is not easily accessible by pulsed electron dipolar spectroscopy. The 19F ENDOR approach relies on spin labeling, and in this work, we compare various labels' performance. Four protein variants of GB1 and ubiquitin bearing fluorinated residues were labeled at the same site with nitroxide and trityl radicals and a Gd(III) chelate. Additionally, a double-histidine variant of GB1 was labeled with a Cu(II) nitrilotriacetic acid chelate. ENDOR measurements were carried out at W-band (95 GHz) where 19F signals are well separated from 1H signals. Differences in sensitivity were observed, with Gd(III) chelates providing the highest signal-to-noise ratio. The new trityl label, OXMA, devoid of methyl groups, exhibited a sufficiently long phase memory time to provide an acceptable sensitivity. However, the longer tether of this label effectively reduces the maximum accessible distance between the 19F and the Cα of the spin-labeling site. The nitroxide and Cu(II) labels provide valuable additional geometric insights via orientation selection. Prediction of electron-nuclear distances based on the known structures of the proteins were the closest to the experimental values for Gd(III) labels, and distances obtained for Cu(II) labeled GB1 are in good agreement with previously published NMR results. Overall, our results offer valuable guidance for selecting optimal spin labels for 19F ENDOR distance measurement in proteins.

Simultaneous Localization of Two High Affinity Divalent Metal Ion Binding Sites in the Tetracycline RNA Aptamer with Mn2+-Based Pulsed Dipolar EPR Spectroscopy.

Mg2+ ions play an essential part in stabilizing the tertiary structure of nucleic acids. While the importance of these ions is well documented, their localization and elucidation of their role in the structure and dynamics of nucleic acids are often challenging. In this work, pulsed electron-electron double resonance spectroscopy (PELDOR, also known as DEER) was used to localize two high affinity divalent metal ion binding sites in the tetracycline RNA aptamer with high accuracy. For this purpose, the aptamer was labeled at different positions with a semirigid nitroxide spin label and diamagnetic Mg2+ was replaced with paramagnetic Mn2+, which did not alter the folding process or ligand binding. Out of the several divalent metal ion binding sites that are known from the crystal structure, two binding sites with high affinity were detected: one that is located at the ligand binding center and another at the J1/2 junction of the RNA.

Product analog binding identifies the copper active site of particulate methane monooxygenase.

Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the CuB, CuC, and CuD sites. Challenges associated with the isolation of active pMMO have hindered progress toward locating its catalytic center. However, reconstituting pMMO into native lipid nanodiscs stabilizes its structure and recovers its activity. Here, these active samples were incubated with 2,2,2,-trifluoroethanol (TFE), a product analog that serves as a readily visualized active-site probe. Interactions of TFE with the CuD site were observed by both pulsed ENDOR spectroscopy and cryoEM, implicating CuD and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoEM maps.

A nitroxide-containing cathode material for organic radical batteries studied with pulsed EPR spectroscopy

An electron spin echo in a nitroxide-containing polymer cathode film for organic radical batteries is observed for various states of charge at cryogenic temperatures. The EPR-detected state of charge (ESOC), as inferred from the number of paramagnetic centers in the film, is compared to the results of Coulomb counting based on galvanostatic charging. Spin concentration, longitudinal relaxation times T1 and phase memory times Tm strongly correlate with the ESOC. In the discharged film, the spin concentration reaches 5±3×1020 cm−3, causing a phase memory time Tm≪ 100 ns (shorter than the resonator ring-down time) that hinders the detection of the spin echo. In the charged film, the decreased spin concentration results in a longer Tm between 100 ns and 300 ns that enables spin-echo detection, yet limits the length of the microwave pulse sequence. The short, broad-band pulses cause instantaneous diffusion in the unoxidized domains across the oxidized film, affecting the relative peak intensities in the pulsed EPR spectrum. By simulating the spectral distortion caused by instantaneous diffusion, we obtain information on the local spin concentration, which complements the information on the ‘bulk’ spin concentration determined by electrochemistry and continuous-wave EPR spectroscopy.

Quantitative polarization-sensitive super-resolution solid immersion microscopy reveals biological tissues’ birefringence in the terahertz range

Terahertz (THz) technology offers a variety of applications in label-free medical diagnosis and therapy, majority of which rely on the effective medium theory that assumes biological tissues to be optically isotropic and homogeneous at the scale posed by the THz wavelengths. Meanwhile, most recent research discovered mesoscale (sim lambda ) heterogeneities of tissues; lambda is a wavelength. This posed a problem of studying the related scattering and polarization effects of THz-wave–tissue interactions, while there is still a lack of appropriate tools and instruments for such studies. To address this challenge, in this paper, quantitative polarization-sensitive reflection-mode THz solid immersion (SI) microscope is developed, that comprises a silicon hemisphere-based SI lens, metal-wire-grid polarizer and analyzer, a continuous-wave 0.6 THz (lambda = 500 µm) backward-wave oscillator (BWO), and a Golay detector. It makes possible the study of local polarization-dependent THz response of mesoscale tissue elements with the resolution as high as 0.15 lambda . It is applied to retrieve the refractive index distributions over the freshly-excised rat brain for the two orthogonal linear polarizations of the THz beam, aimed at uncovering the THz birefringence (structural optical anisotropy) of tissues. The most pronounced birefringence is observed for the Corpus callosum, formed by well-oriented and densely-packed axons bridging the cerebral hemispheres. The observed results are verified by the THz pulsed spectroscopy of the porcine brain, which confirms higher refractive index of the Corpus callosum when the THz beam is polarized along axons. Our findings highlight a potential of the quantitative polarization THz microscopy in biophotonics and medical imaging.

Diffusion of muonic hydrogen in hydrogen gas and the measurement of the 1$s$ hyperfine splitting of muonic hydrogen

The CREMA collaboration is pursuing a measurement of the ground-state hyperfine splitting (HFS) in muonic hydrogen (\muμp) with 1 ppm accuracy by means of pulsed laser spectroscopy. In the proposed experiment, the \muμp atom is excited by a laser pulse from the singlet to the triplet hyperfine sub-levels, and is quenched back to the singlet state by an inelastic collision with a H_22 molecule. The resulting increase of kinetic energy after this cycle modifies the \muμp atom diffusion in the hydrogen gas and the arrival time of the \muμp atoms at the target walls. This laser-induced modification of the arrival times is used to expose the atomic transition. In this paper we present the simulation of the \muμp diffusion in the H_22 gas which is at the core of the experimental scheme. These simulations have been implemented with the Geant4 framework by introducing various low-energy processes including the motion of the H_22 molecules, i.e. the effects related with the hydrogen target temperature. The simulations have been used to optimize the hydrogen target parameters (pressure, temperatures and thickness) and to estimate signal and background rates. These rates allow to estimate the maximum time needed to find the resonance and the statistical accuracy of the spectroscopy experiment.

Time-Resolved Mn2+ -NO and NO-NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter.

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn2+ -NO, and Mn2+ -Mn2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+ -ATP, and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.

Spectroscopically Orthogonal Spin Labels in Structural Biology at Physiological Temperatures.

Electron paramagnetic resonance spectroscopy (EPR) is mostly used in structural biology in conjunction with pulsed dipolar spectroscopy (PDS) methods to monitor interspin distances in biomacromolecules at cryogenic temperatures both in vitro and in cells. In this context, spectroscopically orthogonal spin labels were shown to increase the information content that can be gained per sample. Here, we exploit the characteristic properties of gadolinium and nitroxide spin labels at physiological temperatures to study side chain dynamics via continuous wave (cw) EPR at X band, surface water dynamics via Overhauser dynamic nuclear polarization at X band and short-range distances via cw EPR at high fields. The presented approaches further increase the accessible information content on biomolecules tagged with orthogonal labels providing insights into molecular interactions and dynamic equilibria that are only revealed under physiological conditions.

Comparison of two different ceramic electrodes based on Sb-SnO2 coated with BiFeO3 and Bi2WO6 for the photoelectrooxidation of an emerging pollutant

In this work, a comparison between novel photoanodes based on Sb-SnO2 (BCE) coated with BiFeO3 (BFO-BCE) and Bi2WO6 (BWO-BCE) was carried out. An improvement in the catalytic activity of the electrodes under light exposure was demonstrated by means of Linear Sweep Voltammetry, light pulsed chronoamperometry and Electrochemical Impedance Spectroscopy, being more notorious at current densities below 25 mA·cm−2 for the BFO-BCE and above 25 mA·cm−2 for the BWO-BCE. This improved performance was caused by an increase of the photogenerated oxidizing species. As compared with the uncoated BCE anode used without light, photoelectrooxidation tests led to improvements of around 40% in the degradation degree of norfloxacin (NOR) using both photoanodes at 8.33 mA·cm−2. This improvement was also observed in the mineralization degree of the model wastewaters, with an increase of 36% and 28% at 25 mA·cm−2 for the BWO-BCE and BFO-BCE, respectively. The degradation and formation of subproducts was followed by ion chromatography and HPLC analysis, where some of the main intermediates were detected, allowing us to elaborate a degradation route for NOR with these novel electrodes. The Mineralization Current Efficiency (MCE), energy consumption and extent of electrochemical combustion (Φ) showed improvements with light application for both electrodes at high current densities, being the BWO-BCE the one with the highest MCE and Φ at the cost of a slightly higher energy consumption. This showed the importance of light for these electrodes and its impact in the general process performance, which can be of great advantage in future applications.

Inorganic Chemistry in Dalian University of Technology: Fundamental Science and Solution for Renewable Energy, Environment, Health, and Materials

Inorganic Chemistry in Dalian University of Technology: Fundamental Science and Solution for Renewable Energy, Environment, Health, and Materials

Bayesian optimization to estimate hyperfine couplings from 19F ENDOR spectra

ENDOR spectroscopy is a fundamental method to detect nuclear spins in the vicinity of paramagnetic centers and their mutual hyperfine interaction. Recently, site-selective introduction of 19F as nuclear labels has been proposed as a tool for ENDOR-based distance determination in biomolecules, complementing pulsed dipolar spectroscopy in the range of angstrom to nanometer. Nevertheless, one main challenge of ENDOR still consists of its spectral analysis, which is aggravated by a large parameter space and broad resonances from hyperfine interactions. Additionally, at high EPR frequencies and fields (⩾94 GHz/3.4 Tesla), chemical shift anisotropy might contribute to broadening and asymmetry in the spectra. Here, we use two nitroxide-fluorine model systems to examine a statistical approach to finding the best parameter fit to experimental 263 GHz 19F ENDOR spectra. We propose Bayesian optimization for a rapid, global parameter search with little prior knowledge, followed by a refinement by more standard gradient-based fitting procedures. Indeed, the latter suffer from finding local rather than global minima of a suitably defined loss function. Using a new and accelerated simulation procedure, results for the semi-rigid nitroxide-fluorine two and three spin systems lead to physically reasonable solutions, if minima of similar loss can be distinguished by DFT predictions. The approach also delivers the stochastic error of the obtained parameter estimates. Future developments and perspectives are discussed.

Response surface method optimization of synthesized superparamagnetic magnetite (Fe3O4) nanoparticles characterized by pulsed IR calibration-free laser-induced breakdown spectroscopy

Response surface method optimization of synthesized superparamagnetic magnetite (Fe3O4) nanoparticles characterized by pulsed IR calibration-free laser-induced breakdown spectroscopy

Moisture adsorption by porous terahertz optical materials: a case study of artificial SiO2 opals

Artificial opals fabricated by sedimentation and self-assembly of colloidal SiO2 nanoparticles and annealed at different temperatures were recently considered favorable terahertz (THz) optical materials with manageable optical properties. However, interactions between such a porous material and water vapour in a humid atmosphere can hamper their THz applications due to the related changes in the material parameters and additional power loss. To quantify such an effect, in this paper, moisture adsorption by artificial SiO2 opals is studied using THz pulsed spectroscopy. Particularly, opals of two kinds were sedimented from the colloidal suspension of 300-nm-diameter SiO2 nanoparticles with different intraglobular structures and porosity. They were annealed at temperatures of 200–800°C aimed at changing their internal structure, porosity, and THz optical properties. Opals were dehydrated in a vacuum and then exposed to a humid atmosphere with 82.0 ± 2.0% relative humidity, while their THz complex dielectric permittivity was evaluated in situ in the 0.5–2.5 THz range. The observed changes in the THz dielectric curves were analyzed using the sum rule and the adsorption kinetics models. Our findings reveal a strong dependence of the THz dielectric response, amount of adsorbed water, and adsorption time constant on the opal type and annealing conditions. This effect has a general character: it can hamper real-live applications of a variety of porous THz optical materials and, thus, should be taken into account during their synthesis.

Stepwise generation of spin-polarized radicals and their reactions in the photolysis of an oxime ester compound as studied by pulsed EPR spectroscopy

To generate spin-polarized free radicals, the photolysis of 1,2-octane-dione, 1-[4-(phenylthio)-2-(o-benzoyloxime)] (OXE01) was investigated using time-resolved electron paramagnetic resonance (TR-EPR) and pulsed EPR. This photolysis proceeds through α-cleavage of the N-O bond of OXE01 to yield iminyl and benzoyloxy radicals, the latter of which is converted to a phenyl radical by decarboxylation. The phenyl radical promptly abstracts hydrogen atoms from the surrounding solvent molecules to produce a solvent dehydrogenated radical (solvent radical). The spin polarization created in the initial OXE01 triplet excited state was preserved during these series of reactions, which enhanced EPR sensitivity. This enabled us to observe the TR-EPR spectra of the intermediate free radicals, except phenyl. The decarboxylation rate of the benzoyloxy radical was measured in various solvents by probing the free induction decay signal of the product solvent radicals using pulsed EPR. This new method for preparing free radicals with spin-polarization was applied to determine the addition reaction rate constants of the cyclohexyl radical, a solvent radical, to olefins using pulsed EPR monitoring of the electron spin echo signals of the radical.

“Store-bought is fine”: Sensitivity considerations using shaped pulses for DEER measurements on Cu(II) labels

The narrow excitation bandwidth of monochromic pulses is a sensitivity limitation for pulsed dipolar spectroscopy on Cu(II)-based measurements. In response, frequency-swept pulses with large excitation bandwidths have been adopted to probe a greater range of the EPR spectrum. However, much of the work utilizing frequency-swept pulses in Cu(II) distance measurements has been carried out on home-built spectrometers and equipment. Herein, we carry out systematic Cu(II) based distance measurements to demonstrate the capability of chirp pulses on commercial instrumentation. More importantly we delineate sensitivity considerations under acquisition schemes that are necessary for robust distance measurements using Cu(II) labels for proteins. We show that a 200 MHz sweeping bandwidth chirp pulse can improve the sensitivity of long-range distance measurements by factors of three to four. The sensitivity of short-range distances only increases slightly due to special considerations for the chirp pulse duration relative to the period length of the modulated dipolar signal. Enhancements in sensitivity also dramatically reduce measurement collection times enabling rapid collection of orientationally averaged Cu(II) distance measurements in under two hours.

Thermally activated transformations of the dielectric response function of mono- and disaccharides in the THz range

The variety of physical and chemical properties of saccharides is determined, in particular, by their isomerism and the variability of their hydrogen bond parameters. We studied the transformation of the vibrational spectra of mono- and disaccharides using pulsed terahertz spectroscopy in a wide temperature range. The stability ranges of saccharides were determined from the temperature dependences of the vibrational spectra and resonance parameters. The revealed difference in frequencies and intensities of vibrational modes of the hydroxyl groups in different saccharides correlate with the parameters of hydrogen bonds. Based on the data over a wide temperature range, it was found that the anharmonicity of vibrational modes appears in the form of coupling between resonances and the formation of wide asymmetric bands. Temperature square dependence of the resonance eigenfrequencies in the disaccharides has been revealed.

High-resolution spectroscopy of a single nitrogen-vacancy defect at zero magnetic field

We report a study of high-resolution microwave spectroscopy of nitrogen-vacancy (NV) centers in diamond crystals at and around zero magnetic field. We observe characteristic splitting and transition imbalance of the hyperfine transitions, which originate from level anti-crossings (LACs) in the presence of a transverse effective field. We use pulsed electron spin resonance spectroscopy to measure the zero-field spectral features of single NV centers for clearly resolving such LACs. To quantitatively analyze the magnetic resonance behavior of the hyperfine spin transitions in the presence of the effective field, we present a theoretical model, which describes the transition strengths under the action of an arbitrarily polarized microwave magnetic field. Our results are of importance for the optimization of the experimental conditions for the polarization-selective microwave excitation of spin-1 systems in zero or weak magnetic fields.