Wave Electron Paramagnetic Resonance Spectroscopy Research Articles

Copper Complexes in Verdigris Painting Mixtures: An Electron Paramagnetic Resonance Characterization

Abstract Copper complexes, also called “copper soaps” when referred to carboxylate compounds, have become a concern in the conservation of historical artefacts. These complexes are suspected to play a significant role in degradation processes such as darkening and cracking of painting layers. Here we propose the Continuous Wave Electron Paramagnetic Resonance spectroscopy (CW-EPR) as a tool for the identification of these species. EPR spectroscopy is a micro-invasive and non-destructive technique that is highly sensitive to paramagnetic species, including radicals, crystal defects, and transition metal ions, e.g., Cu(II) and Fe(III). We have selected the verdigris (copper acetate) pigment combined with many organic binders, e.g., egg tempera and gum Arabic, as mock-up systems because of the known degradation tendency of the copper-based pigment. We would like to demonstrate that a precise analysis of the experimental spectra assisted with the calculus of the main magnetic quantities could lead to a good characterization of the copper complexes (or “soaps”).

Probing the Structural Topology and Dynamic Properties of gp28 Using Continuous Wave Electron Paramagnetic Resonance Spectroscopy.

Lysis of Gram-negative bacteria by dsDNA phages is accomplished through either the canonical holin-endolysin pathway or the pinholin-SAR endolysin pathway. During lysis, the outer membrane (OM) is disrupted, typically by two-component spanins or unimolecular spanins. However, in the absence of spanins, phages use alternative proteins called Disruptin to disrupt the OM. The Disruptin family includes the cationic antimicrobial peptide gp28, which is found in the virulent podophage φKT. In this study, EPR spectroscopy was used to analyze the dynamics and topology of gp28 incorporated into a lipid bilayer, revealing differences in mobility, depth parameter, and membrane interaction among different segments and residues of the protein. Our results indicate that multiple points of helix 2 and helix 3 interact with the phospholipid membrane, while others are solvent-exposed, suggesting that gp28 is a surface-bound peptide. The CW-EPR power saturation data and helical wheel analysis confirmed the amphipathic-helical structure of gp28. Additionally, course-grain molecular dynamics simulations were further used to develop the structural model of the gp28 peptide associated with the lipid bilayers. Based on the data obtained in this study, we propose a structural topology model for gp28 with respect to the membrane. This work provides important insights into the structural and dynamic properties of gp28 incorporated into a lipid bilayer environment.

Site-selective and stochastic spin labelling of neutral water-soluble dietary fibers optimized for electron paramagnetic resonance spectroscopy

Use of spin labels to study structures of polymers has been widely spread in polymer science. However, for the studies of neutral water-soluble dietary fibers (DFs), labelling efficiencies in past studies have only been sufficient for application of continuous wave electron paramagnetic resonance spectroscopy (CW-EPR), but still insufficient for some advanced methods such as pulse EPR. Thus, in this paper, two spin labelling strategies, namely, site-selective mono-spin-labelling and stochastic multi-spin-labelling, were examined on linear cereal β-glucan, as well as linearly branched arabinoxylan and galactomannan. The effects of both methods in DF properties were evaluated. For the mono-labelling pathway, labelling efficiency could reach up to 46 %. In the stochastic labelling strategy, a degree of substitution (DS) up to 150 % could be reached, whereas optimized conditions for this strategy were achieved at DS = 3 % to obtain DFs whose bioactivity properties were still preserved while spin labelling efficiency was still sufficient for CW and pulse EPR experiments.

Influence of hydration on segmental chain librations and dynamical transition in lipid bilayers

Continuous wave electron paramagnetic resonance spectroscopy of chain-labeled phospholipids is used to investigate the effects of hydration on the librational oscillations and the dynamical transition of phospholipid membranes in the low-temperature range 120–270 K. Bilayers of dipalmitoylphostatidiycholine (DPPC) spin-labeled at the first acyl chain segments and at the methyl ends and prepared at full, low, and very low hydration are considered. The segmental mean-square angular amplitudes of librations, 〈α2〉, are larger in the bilayer interior than at the polar/apolar interface and larger in the fully and low hydrated than in the very low hydrated membranes. For chain segments at the beginning of the hydrocarbon region, 〈α2〉-values are markedly restricted and temperature independent in DPPC with the lowest water content, whereas they increase with temperature in the low and fully hydrated bilayers, particularly at the highest temperatures. For chain segments at the chain termini, the librational amplitudes increase progressively, first slowly and then more rapidly with temperature in bilayers at any level of hydration. From the temperature dependence of the mean-square librational amplitude, the dynamical transition is detected around 240 K at the polar/apolar interface in fully and low hydrated DPPC and at around 225 K at the inner hydrocarbon region for bilayers at any hydration condition. At the dynamical transition the bilayers cross low energy barriers of activation energy in the range 10–20 kJ/mol. The results highlight biophysical properties of DPPC bilayers at low-temperature and provide evidence of the effects of the hydration on the dynamical transition in bilayers.

Influence of pH on the Growth and the Local Dynamics of Polyelectrolyte Multilayers

Polyelectrolyte multilayers (PEMs) have been prepared using the strong polycation poly(diallyldimethylammonium chloride) and the weak polyanion poly(ethylene-alt-maleic acid) [P(E-alt-MA)]. The spin label (SL) 4-amino-2,2,6,6-tetramethylpiperidine 1-oxide was covalently attached to the polyanion [SL-P(E-alt-MA)] to monitor changes in the signal intensity as a measure of the layer growth and the line shape of the spectra indicative of the polymer chain mobility obtained by continuous wave electron paramagnetic resonance spectroscopy. The aim of this study was to investigate the influence of the preparation conditions. We show the influence of the pH value on the buildup and the mobility of PEMs with 16 and 17 layers, respectively. In the first part, the spin-labeled polyanion was used in the preparation of each polyanion layer to monitor the growth of the multilayers. For all pH values from 3 to 6 a non-linear growth behavior was obtained at the beginning of the PEM buildup where the surface is not yet fully covered, leading to a linear growth after reaching a transition point. This point depends on the pH value. In a second study, the spin-labeled polyanion was selectively placed in one layer to display changes in the rotational mobility of the SL in this layer within the multilayers. As a result, an increase of the rotational mobility of SL-P(E-alt-MA) with increasing pH value was observed.

Continuous Wave Electron Paramagnetic Resonance Spectroscopy Reveals the Structural Topology and Dynamic Properties of Active Pinholin S2168 in a Lipid Bilayer.

Pinholin S2168 is an essential part of the phage Φ21 lytic protein system to release the virus progeny at the end of the infection cycle. It is known as the simplest natural timing system for its precise control of hole formation in the inner cytoplasmic membrane. Pinholin S2168 is a 68 amino acid integral membrane protein consisting of two transmembrane domains (TMDs) called TMD1 and TMD2. Despite its biological importance, structural and dynamic information of the S2168 protein in a membrane environment is not well understood. Systematic site-directed spin labeling and continuous wave electron paramagnetic resonance (CW-EPR) spectroscopic studies of pinholin S2168 in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) proteoliposomes are used to reveal the structural topology and dynamic properties in a native-like environment. CW-EPR spectral line-shape analysis of the R1 side chain for 39 residue positions of S2168 indicates that the TMDs have more restricted mobility when compared to the N- and C-termini. CW-EPR power saturation data indicate that TMD1 partially externalizes from the lipid bilayer and interacts with the membrane surface, whereas TMD2 remains buried in the lipid bilayer in the active conformation of pinholin S2168. A tentative structural topology model of pinholin S2168 is also suggested based on EPR spectroscopic data reported in this study.

Synthesis and Characterization of Cu-Ni Mixed Metal Paddlewheels Occurring in the Metal-Organic Framework DUT-8(Ni0.98Cu0.02) for Monitoring Open-Closed-Pore Phase Transitions by X-Band Continuous Wave Electron Paramagnetic Resonance Spectroscopy.

A Cu2+-doped metal-organic framework (DUT-8(Ni0.98Cu0.02), M2(NDC)2DABCO, M = Ni, Cu, NDC = 2,6-napththalene dicarboxylate, DABCO = 1,4-diazabicyclo[2.2.2]octane, DUT = Dresden University of Technology) was synthesized in the form of large (>1 μm) and small crystals (<1 μm) to analyze their switchability by X-band continuous wave (cw) electron paramagnetic resonance (EPR) spectroscopy. The large crystals are flexible and in a porous open pore (op) phase after solvation in N, N-dimethylformamide (DMF), but in the activated solvent-free form, a nonporous closed pore (cp) phase forms. EPR measurements of the rigid Ni-free DUT-8(Cu) show a characteristic electron spin S = 1 room temperature signal of the antiferromagnetically coupled Cu2+-Cu2+ paddlewheel building unit of this metal-organic framework. None of the mixed metal DUT-8(Ni0.98Cu0.02) materials showed comparable signals, indicating the absence of dimeric Cu2+-Cu2+ paddlewheel units in the materials. Instead, characteristic electron spin S = 3/2 signals are detected for all DUT-8(Ni0.98Cu0.02) samples at temperatures T < 77 K, which can be assigned to ferromagnetically coupled mixed metal Ni2+-Cu2+ paddlewheel units. Those signals differ characteristically for the op and cp phase and enable monitoring the reversible op-cp transition during the de-/adsorption of DMF.

High-Yield Spin Labeling of Long RNAs for Electron Paramagnetic Resonance Spectroscopy.

Site-directed spin labeling is a powerful tool for investigating the conformation and dynamics of biomacromolecules such as RNA. Here we introduce a spin labeling strategy based on click chemistry in solution that, in combination with enzymatic ligation, allows highly efficient labeling of complex and long RNAs with short reaction times and suppressed RNA degradation. With this approach, a 34-nucleotide aptamer domain of the preQ1 riboswitch and an 81-nucleotide TPP riboswitch aptamer could be labeled with two labels in several positions. We then show that conformations of the preQ1 aptamer and its dynamics can be monitored in the absence and presence of Mg2+ and a preQ1 ligand by continuous wave electron paramagnetic resonance spectroscopy at room temperature and pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) in the frozen state.

Low-Temperature Dynamics of Chain-Labeled Lipids in Ester- and Ether-Linked Phosphatidylcholine Membranes.

Continuous wave electron paramagnetic resonance spectroscopy and two-pulse echo detected spectra of chain-labeled lipids are used to study the dynamics of frozen lipid membranes over the temperature range 77-260 K. Bilayers of ester-linked dihexadecanoylphosphatidylcholine (DPPC) with noninterdigitated chains and ether-linked dihexadecyl phosphatidylcholine (DHPC) with interdigitated chains are considered. Rapid stochastic librations of small angular amplitude are found in both lipid matrices. In noninterdigitated DPPC bilayers, the mean-square angular amplitude, [Formula: see text], of the motion increases with temperature and it is larger close to the chain termini than close to the polar/apolar interface. In contrast, in interdigitated DHPC lamellae, [Formula: see text] is small and temperature and label-position independent at low temperature and increases steeply at high temperature. The rotational correlation time, τc, of librations lies in the subnanosecond range for DPPC and in the nanosecond range for DHPC. In all membrane samples, the temperature dependence of [Formula: see text] resembles that of the mean-square atomic displacement revealed by neutron scattering and a dynamical transition is detected in the range 210-240 K. The results highlight the librational oscillations and the glass-like behavior in bilayer and interdigitated lipid membranes.

Elucidation of Molecular Mechanism Underlying KcsA's Hysteretic Gating Behavior

Hysteresis has been observed in cyclic nucleotide-gated4, transient receptor potential vanilloid3, N-methyl-D-aspartate5, human Ether-a-go-go-Related Gene6, human HCN48, mouse HCN11 channels. Voltage shift for QV (gating charge vs voltage) curve is well documented for voltage-gated cation channels, which was considered an intrinsic property of the voltage-sensing domain (VSD). However, it was showed recently that uncoupling the VSD from the pore domain of the Shaker K+ channel, eliminated the VSD's hysteretic gating behavior. It was suggested that the pore domain imposes a mechanical load on the VSD due to stabilization of an open state, which causes the hysteresis2. Since open-state stabilization occur at the channels’ pore domain, it is important to study the pore domain gating mechanism in isolation. We intend to use the archetypal pore domain of a K+ channel, KcsA, as a functional and structural surrogate, to elucidate the molecular basis of hysteretic gating behavior in ion channels. Recently, we have unveiled a novel hysteretic gating behavior in KcsA by electrophysiology and by continuous wave electron paramagnetic resonance spectroscopy, which faithfully measure the pH dependent conformational changes associated to activation7 and deactivation gating. We hypothesize that structural changes of the KcsA's selectivity filter associated to C-type inactivation underlie the molecular mechanism of hysteretic gating in KcsA and by extension in other ion channels. The long-term goal of this project is to determine the molecular basis for hysteretic gating in ion channels, which can be useful for the development of drugs that can correct several channelopathies.

Structural and electrical properties of metastable defects in hydrogenated amorphous silicon

The structural and electrical properties of metastable defects in various types of hydrogenated amorphous silicon have been studied using a powerful combination of continuous wave electron-paramagnetic resonance spectroscopy, electron spin echo (ESE) decay measurements, and Doppler broadening positron annihilation spectroscopy. The observed dependence of the paramagnetic defect density on the Doppler S parameter indicates that porous, nanosized void-rich materials exhibit higher spin densities, while dense, divacancy-dominated materials show smaller spin densities. However, after light soaking more similar spin densities are observed, indicating a long-term defect creation process in the Staebler-Wronski effect that does not depend on the a-Si:H nanostructure. From ESE decays it appears that there are fast and slowly relaxing defect types, which are linked to various defect configurations in small and large open volume deficiencies. A nanoscopic model for the creation of light-induced defects in the a-Si:H nanostructure is proposed.

Small Substrate Transport and Mechanism of a Molybdate ATP Binding Cassette Transporter in a Lipid Environment

Embedded in the plasma membrane of all bacteria, ATP binding cassette (ABC) importers facilitate the uptake of several vital nutrients and cofactors. The ABC transporter, MolBC-A, imports molybdate by passing substrate from the binding protein MolA to a membrane-spanning translocation pathway of MolB. To understand the mechanism of transport in the biological membrane as a whole, the effects of the lipid bilayer on transport needed to be addressed. Continuous wave-electron paramagnetic resonance and in vivo molybdate uptake studies were used to test the impact of the lipid environment on the mechanism and function of MolBC-A. Working with the bacterium Haemophilus influenzae, we found that MolBC-A functions as a low affinity molybdate transporter in its native environment. In periods of high extracellular molybdate concentration, H. influenzae makes use of parallel molybdate transport systems (MolBC-A and ModBC-A) to take up a greater amount of molybdate than a strain with ModBC-A alone. In addition, the movement of the translocation pathway in response to nucleotide binding and hydrolysis in a lipid environment is conserved when compared with in-detergent analysis. However, electron paramagnetic resonance spectroscopy indicates that a lipid environment restricts the flexibility of the MolBC translocation pathway. By combining continuous wave-electron paramagnetic resonance spectroscopy and substrate uptake studies, we reveal details of molybdate transport and the logistics of uptake systems that employ multiple transporters for the same substrate, offering insight into the mechanisms of nutrient uptake in bacteria.

Hydration layer coupling and cooperativity in phase behavior of stimulus responsive peptide polymers.

It is shown that hydrophilic (backbone) and hydrophobic (side chain) hydration layers of elastin-like polypeptides (ELPs), a class of stimulus responsive peptide polymers that exhibit lower critical solution temperature (LCST) phase transition behavior, can exist in a coupled and decoupled state. The decoupled hydration state consists of hydrophobic and hydrophilic hydration layers that respond independently to temperature, while the coupled hydration state is characterized by a common, cooperative dehydration of both hydration layers. It is further shown that the primary sequence of an ELP can be tuned to exhibit either of the hydration layer coupling modes. Charged side chains lead to decoupling, while strongly hydrophobic side chains trigger stronger interaction between hydrophilic and hydrophobic hydration, leading to coupling of both layers. Further, for aprotic residues this coupling is fostered by decreasing bulkiness of hydrophobic side chains due to larger hydration numbers and water molecules mediating coupling between side chain and backbone hydration shells. For coupled hydration shells, the LCST phase transition characterized by spin probing continuous wave electron paramagnetic resonance spectroscopy is reminiscent of a first-order process even on nanoscopic length scales. In contrast, analogous synthetic polymers exhibit nanoscale phase transitions over a broad temperature range, indicating that their nanoscale phase behavior is not of first order. Hence, our results indicate that ELPs are the first identified class of polymers that exhibit a first-order inverse phase transition on nanoscopic length scales. These results may also provide insights into the role of hydration layers in governing the structure-function relationship of intrinsically disordered proteins.

Monitoring the autoproteolysis of hiv-1 protease by site-directed spin-labeling and electron paramagnetic resonance spectroscopy

Site-directed spin-labeling with continuous wave electron paramagnetic resonance spectroscopy was used to monitor autoproteolysis of HIV-1 protease, an enzyme essential for viral maturation. Two protein constructs were examined, namely subtype F and the circulating recombinant form CRF01_A/E. As the protease undergoes self-cleavage, protein unfolds and small peptide fragments containing the spin label are generated, which collectively give rise to a sharp spectral component that is easily discernable in the high-field resonance line in the EPR spectrum. By monitoring the intensity of this spectral component over time, the autoproteolytic stability of each construct was characterized under various conditions. Data were collected for samples stored at 4 °C, 25 °C, and 37 °C, and on a subtype F HIV-1 protease sample stored at 25 °C and containing the FDA-approved protease inhibitor Tipranavir. As expected, the rate of autoproteolysis decreased as the storage temperature was lowered. Minimal autoproteolysis was seen for the sample that contained Tipranavir, providing direction for future spectroscopic studies of active protease samples. When compared to standard methods of monitoring protein degradation such as gel electrophoresis or chromatographic analyses, spin-labeling with CW EPR offers a facile, real-time, non-consuming way to monitor autoproteolysis or protein degradation. Additionally, mass spectrometry studies revealed that the N-termini of both constructs are sensitive to degradation and that the sites of specific autoproteolysis vary.

Formation of the Complex of Nitrite with the Ferriheme b β-Barrel Proteins Nitrophorin 4 and Nitrophorin 7,

The interaction of ferriheme proteins with nitrite has recently attracted interest as a source for NO or other nitrogen oxides in mammalian physiology. However, met-hemoglobin (metHb), which was suggested as a key player in this process, does not convert nitrite unless small amounts of NO are added in parallel. We have recently reported that, in contrast, nitrophorins (NPs) convert nitrite as the sole substrate to form NO even at pH 7.5, which is an unprecedented case among ferrihemes [He, C., and Knipp, M. (2009) J. Am. Chem. Soc. 131, 12042-12043]. NPs, which comprise a class of unique heme b proteins from the saliva of the blood-sucking insect Rhodnius prolixus, appear in a number of concomitant isoproteins. Herein, the first spectroscopic characterization of the initial complexes of the two isoproteins NP4 and NP7 with nitrite is presented and compared to the data reported for metHb and met-myoglobin (metMb). Because upon nitrite binding, NPs, in contrast to metHb and metMb, continue to react with nitrite, resonance Raman spectroscopy and continuous wave electron paramagnetic resonance spectroscopy were applied to frozen samples. As a result, the existence of two six-coordinate ferriheme low-spin complexes was established. Furthermore, X-ray crystallography of NP4 crystals soaked with nitrite revealed the formation of an eta(1)-N nitro complex, which is in contrast to the eta(1)-O-bound nitrite in metMb and metHb. Stopped-flow kinetic experiments show that although the ligand dissociation constants of NP4 and NP7 (15-190 M(-1)) are comparable to those of metHb and metMb, the rates of ligand binding and release are significantly slower. Moreover, not only the reaction kinetics but also electron paramagnetic resonance spectroscopy reveals notable differences between the two isoproteins.

Stacked Sets of Parallel, In-register β-Strands of β2-Microglobulin in Amyloid Fibrils Revealed by Site-directed Spin Labeling and Chemical Labeling

β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.

Cryogenic Q-band (35 GHz) probehead featuring large excitation microwave fields for pulse and continuous wave electron paramagnetic resonance spectroscopy: Performance and applications

The construction and performance of a Q-band (35 GHz) cryogenic probehead for pulse electron paramagnetic resonance and continuous wave electron paramagnetic resonance measurements with down-scaled loop gap resonators (LGRs) is presented. The advantage of the LGR in comparison to TE 012 resonators lies in the large B 1 microwave (mw) fields that can be generated with moderate input mw power. We demonstrated with several examples that this allows optimal performance for double-quantum electron coherence, HYSCORE, and hyperfine decoupling experiments employing matched and high turning angle mw pulses with high B 1-fields. It is also demonstrated that with very low excitation power (i.e. 10–40 mW), B 1-fields in LGRs are still sufficient to allow short mw pulses and thus experiments such as HYSCORE with high-spin systems to be performed with good sensitivity. A sensitivity factor Λ rs of LGRs with different diameters and lengths is introduced in order to compare the sensitivity of different resonant structures. The electromagnetic field distribution, the B 1-field homogeneity, the E 1-field strength, and the microwave coupling between wave guide and LGRs are investigated by electromagnetic field calculations. The advantage and application range using LGRs for small sample diameters is discussed.

Continuous wave electron paramagnetic resonance spectroscopy in the investigation of the surface properties and chemical reactivity of an ionic oxide (MgO)

The surface reactivity of metal oxides has been studied by continuous wave electron paramagnetic resonance spectroscopy and is discussed by means of selected examples concerning magnesium oxide. In particular, the influence of external physical or chemical treatments experienced by the solid on its surface structure and reactivity is illustrated. Examples are given of the effects of progressive dehydration of the solid, doping with transition metal ions and, particularly, addition of alkali metal vapours.

Triplet spin alignment of “stretched porphycenes”

The photoexcited triplet states of 22π and 26π acetylene—cumulene porphycenes (“stretched porphycenes”) were investigated by time-resolved continuous wave electron paramagnetic resonance spectroscopy. Experiments were carried out in isotropic (toluene) and anisotropic (a nematic liquid crystal) matrices. These porphyrinoid compounds exhibit a red-shift in the absorption spectra as a function of molecular length and compared to the 18π porphycene. On the other hand, the zero-field splitting parameter, D, does not exhibit a dependence on the molecular length. This anomaly is interpreted in terms of the sign and magnitude of D, resulting from triplet spin alignment in the molecular plane.

Determination of the position of the Qi− quinone binding from the protein surface of the cytochrome bc1 complex in Rhodobacter capsulates chromatophores

The technique of distance measurement utilizing spin relaxation enhancement by an external probe has been extended to the study of intrinsic semiquinone radicals through the use of holmium-EDTA complexes and continuous wave electron paramagnetic resonance spectroscopy. This technique has been used to determine the distance of the semiquinone anion. Q 1 (also designate as Q n − or Q c − ), from the surface of the ubiquinone cytochrome c oxidoreductase, consisting of only three subunits, in membrane particles from Rhodobacter capsulates . The location of the semiquinone anion is 6–10Åfrom the N side protein, establishing that there are two separate quinone reaction sites, i.e., ‘Q 1 ’ and ‘Q 0 ’, within this complex on opposite sides of the membrane. The results are discussed in relation to reported ENDOR, EPR, and optical studies of the mitochondrial counterpart.