Encapsulated Atomic Hydrogen in Octamethyl-POSS cages: a Pulsed EPR Study.

Encapsulated atomic hydrogen in polyhedral oligomeric silsesquioxane (POSS) cages is a promising candidate for spin-based quantum technologies. Key parameters such as spin relaxation times and magnetic interactions with surrounding electron and nuclear spins can be typically probed with advanced electron paramagnetic resonance (EPR) methods. Here we present a detailed pulsed EPR study of the species H@Si8O12R8 with R=CH3, namely encapsulated atomic hydrogen in the octamethyl POSS derivative. The temperature dependence of the spin-lattice relaxation rate 1/T1 is analyzed in terms of a Raman process with a Debye temperature of ΘD=145 K and a thermally activated process with Ea=794 K (552 cm-1), whereas, the phase memory time TM shows the typical shortening behaviour at T<150 K observed for all methyl-containing derivatives. The hyperfine coupling of the cage 29Si nuclei is measured by hyperfine sublevel correlation (HYSCORE) spectroscopy and is found to fulfil the so-called "matching condition" at the low-field EPR transition. The potential of this paramagnetic molecule to perform one-qubit quantum operations is probed by room-temperature Rabi oscillations.

Spin-Labeled Diclofenac: Synthesis and Interaction with Lipid Membranes.

Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) from the group of phenylacetic acid derivatives, which has analgesic, anti-inflammatory and antipyretic properties. The interaction of non-steroidal anti-inflammatory drugs with cell membranes can affect their physicochemical properties, which, in turn, can cause a number of side effects in the use of these drugs. Electron paramagnetic resonance (EPR) spectroscopy could be used to study the interaction of diclofenac with a membrane, if its spin-labeled analogs existed. This paper describes the synthesis of spin-labeled diclofenac (diclofenac-SL), which consists of a simple sequence of transformations such as iodination, esterification, Sonogashira cross-coupling, oxidation and saponification. EPR spectra showed that diclofenac-SL binds to a lipid membrane composed of palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). 2H electron spin echo spectroscopy (ESEEM) was used to determine the position of the diclofenac-SL relative to the membrane surface. It was established that its average depth of immersion corresponds to the 5th position of the carbon atom in the lipid chain.

Electron and nuclear magnetic properties near ZEFOZ region.

In the vicinity of spin level anti-crossings, electron-nuclear spin systems reveal characteristic features that have been investigated by electron paramagnetic resonance (EPR) methods, including electron spin echo envelope modulation (ESEEM). The spectral properties depend considerably on the difference, ΔB, between the magnetic field and the critical field at which the zero first-order Zeeman shift (ZEFOZ) occurs. To analyze the characteristic features near the ZEFOZ point, analytical expressions for the behavior of EPR spectra and ESEEM traces as a function of ΔB are obtained. It is shown that the influence of hyperfine interactions (HFI) decreases linearly when approaching the ZEFOZ point. The HFI splitting of the EPR lines is essentially independent of ΔB near the ZEFOZ point, while the depth of the ESEEM signal has an approximately quadratic dependence on ΔB with a small cubic asymmetry due to the Zeeman interaction of the nuclear spin.

Probing Methyl Group Tunneling in [(CH3)2NH2][Zn(HCOO)3] Hybrid Perovskite Using Co2+ EPR.

At low temperature, methyl groups act as hindered quantum rotors exhibiting rotational quantum tunneling, which is highly sensitive to a local methyl group environment. Recently, we observed this effect using pulsed electron paramagnetic resonance (EPR) in two dimethylammonium-containing hybrid perovskites doped with paramagnetic Mn2+ ions. Here, we investigate the feasibility of using an alternative fast-relaxing Co2+ paramagnetic center to study the methyl group tunneling, and, as a model compound, we use dimethylammonium zinc formate [(CH3)2NH2][Zn(HCOO)3] hybrid perovskite. Our multifrequency (X-, Q- and W-band) EPR experiments reveal a high-spin state of the incorporated Co2+ center, which exhibits fast spin-lattice relaxation and electron spin decoherence. Our pulsed EPR experiments reveal magnetic field independent electron spin echo envelope modulation (ESEEM) signals, which are assigned to the methyl group tunneling. We use density operator simulations to extract the tunnel frequency of 1.84 MHz from the experimental data, which is then used to calculate the rotational barrier of the methyl groups. We compare our results with the previously reported Mn2+ case showing that our approach can detect very small changes in the local methyl group environment in hybrid perovskites and related materials.

Changes in the Electron Transfer Symmetry in the Photosystem I Reaction Centers upon Removal of Iron-Sulfur Clusters.

In photosynthetic reaction centers of intact photosystem I (PSI) complexes from cyanobacteria, electron transfer at room temperature occurs along two symmetrical branches of redox cofactors A and B at a ratio of ~3 : 1 in favor of branch A. Previously, this has been indirectly demonstrated using pulsed absorption spectroscopy and more directly by measuring the decay modulation frequencies of electron spin echo signals (electron spin echo envelope modulation, ESEEM), which allows to determine the distance between the separated charges of the primary electron donor P700+ and phylloquinone acceptors A1A- and A1B- in the symmetric redox cofactors branches A and B. In the present work, these distances were determined using ESEEM in PSI complexes lacking three 4Fe-4S clusters, FX, FA, and FB, and the PsaC protein subunit (the so-called P700-A1 core), in which phylloquinone molecules A1A and A1B serve as the terminal electron acceptors. It was shown that in the P700-A1 core preparations, the average distance between the centers of the P700+A1- ion-radical pair at a temperature of 150 K in an aqueous glycerol solution and in a dried trehalose matrix, as well as in a trehalose matrix at 280 K, is ~25.5 Å, which corresponds to the symmetrical electron transfer along the A and B branches of redox cofactors at a ratio of 1 : 1. Possible reasons for the change in the electron transfer symmetry in PSI upon removal of the PsaC subunit and 4Fe-4S clusters FX, FA, and FB are discussed.

OOP-ESEEM Spectroscopy: Accuracies of Distances of Spin-Correlated Radical Pairs in Biomolecules.

In addition to the commonly used electron-electron double resonance (ELDOR) technique, there are several other electron paramagnetic resonance (EPR) methods by which structure information can be obtained by exploiting the dipolar coupling between two radicals based on its characteristic r -3 dependence. In this contribution, we explore the potential of out-of-phase-electron-spin echo envelope modulation (OOP-ESEEM) spectroscopy to collect accurate distance information in photo-sensitive (bio) molecules. Although the method has already been applied to spin-correlated radical pairs in several classes of light-active proteins, the accuracy of the information obtained has not yet been extensively evaluated. To do this in a system-independent fashion, OOP-ESEEM time traces simulated with different values of the dipolar and exchange couplings were generated and analyzed in a best-possible way. Excellent agreement between calculated and numerically fitted values over a wide range of distances (between 15 and 45 Å) was obtained. Furthermore, the limitations of the method and the dependence on various experimental parameters could be evaluated.

Synthesis of Spin-Labeled Ibuprofen and Its Interaction with Lipid Membranes.

Ibuprofen is a non-steroidal anti-inflammatory drug possessing analgesic and antipyretic activity. Electron paramagnetic resonance (EPR) spectroscopy could be applied to study its interaction with biological membranes and proteins if its spin-labeled analogs were synthesized. Here, a simple sequence of ibuprofen transformations-nitration, esterification, reduction, Sandmeyer reaction, Sonogashira cross-coupling, oxidation and saponification-was developed to attain this goal. The synthesis resulted in spin-labeled ibuprofen (ibuprofen-SL) in which the spin label TEMPOL is attached to the benzene ring. EPR spectra confirmed interaction of ibuprofen-SL with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers. Using 2H electron spin echo envelope modulation (ESEEM) spectroscopy, ibuprofen-SL was found to be embedded into the hydrophobic bilayer interior.

Paramagnetic resonance investigation of mono- and di-manganese-containing systems in biochemistry.

Although not as important as iron, manganese is one of the key transition metal (d block) elements in biochemistry. The role of Mn in photosynthesis is perhaps its "starring role," but has been extensively covered elsewhere and will not be discussed in this chapter. In contrast to iron, for which 57Fe MÓ§ssbauer effect (nuclear gamma resonance) spectroscopy is available, 55Mn (100% abundant) lacks this ability. Instead, the defining technique for Mn is electron paramagnetic resonance (EPR) spectroscopy, as Mn(II), Mn(IV), and even Mn(III), as well as certain di-Mn systems are generally EPR active. This chapter describes how EPR and its advanced derivatives, electron spin echo envelope modulation (ESEEM) and electron-nuclear/electron-electron double resonance (ENDOR/ELDOR) spectroscopies, provide valuable biochemical information on many Mn-containing systems. These include endogenous Mn in mono- and di-Mn (protein) enzymes as well as brief mention of applications to Mn in non-protein biological systems.

Spectroscopic characterization of Mn2+ and Cd2+ coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme.

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-SRp substitution is inhibitory in Mg2+, thiophilic Cd2+ rescues HHRz activity. Mn2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-SRp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn2+ and Cd2+ coordination to Rp and Sp phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd2+ does indeed bind to S in the thio-substituted ligand, Mn2+ coordinates to the non­sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of MnO over MnS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn2+ coordination preference of oxygen in either Rp or Sp A9 phosphorothioates differentially tunes catalytic activity, with MnO coordination in the A9-SRp phosphorothioate enzyme being inhibitory.

Probing the local secondary structure of bacteriophage S21 pinholin membrane protein using electron spin echo envelope modulation spectroscopy.

There have recently been advances in methods for detecting local secondary structures of membrane protein using electron paramagnetic resonance (EPR). A three pulsed electron spin echo envelope modulation (ESEEM) approach was used to determine the local helical secondary structure of the small hole forming membrane protein, S21 pinholin. This ESEEM approach uses a combination of site-directed spin labeling and 2H-labeled side chains. Pinholin S21 is responsible for the permeabilization of the inner cytosolic membrane of double stranded DNA bacteriophage host cells. In this study, we report on the overall global helical structure using circular dichroism (CD) spectroscopy for the active form and the negative-dominant inactive mutant form of S21 pinholin. The local helical secondary structure was confirmed for both transmembrane domains (TMDs) for the active and inactive S21 pinholin using the ESEEM spectroscopic technique. Comparison of the ESEEM normalized frequency domain intensity for each transmembrane domain gives an insight into the α-helical folding nature of these domains as opposed to a π or 310-helix which have been observed in other channel forming proteins.

Protein and solutes freeze-concentration in water/glycerol mixtures revealed by pulse EPR.

Lyophilization can extend protein drugs stability and shelf life, but it also can lead to protein degradation in some cases. The development of safe freeze-drying approaches for sensitive proteins requires a better understanding of lyophilization on the molecular level. The evaluation of the freezing process and its impact on the protein environment in the nm scale is challenging because feasible experimental methods are scarce. In the present work we apply pulse EPR as a tool to study the local concentrations of the solute in the 20 nm range and of the solvent in the 1 nm range for a spin labeled 27 kDa monomeric green fluorescent protein, mEGFP, and the 172 Da TEMPOL spin probe, frozen in different water/glycerol-d5 mixtures. For average glycerol volume fractions, φgly-d5avg, ≥ 0.4 we observed transparent glassy media; the local concentration and the 1 nm solvent shell of TEMPOL and the protein correspond to those of a uniform vitrified glass. At φgly-d5avg ≤ 0.3 we observed partial ice crystallization, which led to ice exclusion of glycerol and TEMPOL with freeze-concentration up to the glycerol maximal-freeze local volume fraction, φgly-d5loc, of 0.64. The protein concentration and its shell behavior was similar except for the lowest φgly-d5avg (0.1), which showed a 4.7-fold freeze-concentration factor compared to sevenfold for TEMPOL, and also a smaller φgly-d5loc. We explain this behavior with an increased probability for proteins to get stuck in the ice phase during fast freezing at higher freeze-concentration and the related large-scale mass transfer.

Tylopeptin B peptide antibiotic in lipid membranes at low concentrations: Self-assembling, mutual repulsion and localization.

The medium-length peptide Tylopeptin B possesses activity against Gram-positive bacteria. It binds to bacterial membranes altering their mechanical properties and increasing their permeability. This action is commonly related with peptide self-assembling, resulting in the formation of membrane channels. Here, pulsed double electron-electron resonance (DEER) data for spin-labeled Tylopeptin B in palmitoyl-oleoyl-glycero-phosphocholine (POPC) model membrane reveal that peptide self-assembling starts at concentration as low as 0.1 mol%; above 0.2 mol% it attains a saturation-like dependence with a mean number of peptides in the cluster = 3.3. Using the electron spin echo envelope modulation (ESEEM) technique, Tylopeptin B molecules are found to possess a planar orientation in the membrane. In the peptide concentration range between 0.1 and 0.2 mol%, DEER data show that the peptide clusters have tendency of mutual repulsion, with a circle of inaccessibility of radius around 20 nm. It may be proposed that within this radius the peptides destabilize the membrane, providing so the peptide antimicrobial activity. Exploiting spin-labeled stearic acids as a model for free fatty acids (FFA), we found that at concentrations of 0.1-0.2 mol% the peptide promotes formation of lipid-mediated FFA clusters; further increase in peptide concentration results in dissipation of these clusters.

Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy.

Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein labeling; and utilizing Cu(II)NTA labeled proteins, to measure distance constraints and backbone dynamics. In doing so, EPR measurements are less influenced by sidechain motion, which influences the breadth of the measured distance distributions between two spins, as well as the measured residue-specific dynamics. More broadly, such EPR-based distance measurements provide unique structural constraints for integrative structural biophysics and complement traditional biophysical techniques, such as NMR, cryo-EM, FRET, and crystallography. Graphic abstract: Monitoring the success of Cu(II)NTA labeling.

Buffer effects on site directed Cu2+-labeling using the double histidine motif.

The double histidine, or dHis, motif has emerged as a powerful spin labeling tool to determine the conformations and dynamics, subunit orientation, native metal binding site location, and other physical characteristics of proteins by Cu2+-based electron paramagnetic resonance. Here, we investigate the efficacy of this technique in five common buffer systems, and show that buffer choice can impact the loading of Cu2+-NTA into dHis sites, and more generally, the sensitivity of the overall technique. We also present a standardized and optimized examination of labeling of the dHis motif with Cu2+-NTA for EPR based distance measurements. We provide optimal loading procedures, using representative EPR and UV/Vis data for each step in the process. From this data, we find that maximal dHis loading can be achieved in under 30 min with low temperature sample incubation. Using only these optimal procedures, we see up to a 28% increase in fully labeled proteins compared to previously published results in N-ethylmorpholine. Using both this optimized procedure as well as a more optimal buffer, we can achieve up to 80% fully loaded proteins, which corresponds to a 64% increase compared to the prior data. These results provide insight and deeper understanding of the dHis Cu2+-NTA system, the variables that impact its efficacy, and present a method by which these issues may be mitigated for the most efficient labeling.

Cryogenically frozen PEGylated liposomes and micelles: Water penetration and polarity profiles.

Poly(ethylene glycol) (PEG)-grafted lipid dispersions are widely investigated in fundamental and biotechnological research for their successful use in drug-delivery. Here, we consider mixtures of the bilayer-forming lipid dipalmitoylphosphatidylcholine (DPPC) with the micelle-forming lipid PEG:2000-phosphatidilethanolamine (PEG:2000-DPPE) fully hydrated in D2O and measured at 77 K. Electron Spin Echo Envelope Modulation and continuous wave Electron Paramagnetic Resonance of chain-labelled lipids are employed to detect the extent of solvent permeation and the environmental polarity, respectively, across the hydrocarbon regions of the lipid assemblies. Sigmoidal water penetration and polarity profiles are described in sterically stabilized liposomes (SSL) formed at submicellar content of PEG:2000-DPPE incorporated in DPPC. Compared to DPPC bilayers, SSL show increased hydrophobicity at both the polar/apolar interface and the chain termini, and a broader transition that is shifted toward the interface. Solvent exposure and polarity decrease on going down the chain in PEG:2000-DPPE micelles. However, compared to SSL, polymer-lipid micelles show higher solvent permeation at any chain segment and the chain termini are accessible to water. In any sample, heterogeneity is found in H-bond formation between the spin-label nitroxide groups and the solvent molecules. The results at cryogenic temperature add new insights into the biophysico-chemical characterization of PEGylated lipid dispersions.

Determining the Electronic Structure of Paramagnetic Intermediates in membrane proteins: A high-resolution 2D 1H hyperfine sublevel correlation study of the redox-active tyrosines of photosystem II.

The photosynthetic reaction center, photosystem II (PSII), catalyzes one of the most energetically demanding reactions in nature by using light energy to drive water oxidation. The four-electron water oxidation reaction occurs at the tetranuclear manganese­calcium-oxo (Mn4Ca-oxo) cluster that is present in the oxygen-evolving complex (OEC) of PSII. The water oxidation reaction is facilitated by proton-coupled electron transfer (PCET) at the redox-active tyrosine residue, YZ, in the OEC which is one of the two symmetric tyrosine residues, YZ and YD, in PSII. Although YZ and YD are chemically identical, their redox properties and reaction kinetics are very different. In the present study, we apply high-resolution two-dimensional (2D) 1H hyperfine sublevel correlation (HYSCORE) spectroscopy to determine the electronic structure of YZ and YD to understand better the functional tuning of PCET at each tyrosine. Most importantly, the 2D HYSCORE measurements that are described here are applicable for the study of paramagnetic cofactors in a wide variety of membrane-bound proteins.

Molecular Structure of Binary Chromium(III)-DNA Adducts.

Chromium(VI) is a carcinogen and mutagen, and its mechanisms of action are proposed to involve binding of its reduction product, chromium(III), to DNA. The manner in which chromium(III) binds DNA has not been established, particularly at a molecular level. Analysis of oligonucleotide duplex DNAs by NMR, EPR, and IR spectroscopies in the presence of chromium(III) allows the elucidation of the Cr binding site. The metal centers were found to interact exclusively with guanine N7 positions. No evidence of chromium interactions with other bases or backbone phosphates nor of Cr forming intra-strand crosslinks between neighboring guanine residues was observed.

Solvent accessibility in interdigitated and micellar phases formed by DPPC/Lyso-PPC mixtures: D2O-ESEEM of chain labeled lipids.

Electron spin echo envelope modulation (ESEEM) spectroscopy was used to investigate binary mixtures of single-chain micelle-forming lipids and diacyl bilayer-forming lipids dispersed in D2O at 77 K. Mixtures of dipalmitoylphosphatidylcholine (DPPC) and lyso-palmitoylphosphatidylcholine (Lyso-PPC) over the entire composition range (0-100 mol%) and phosphatidylcholine spin-labeled at selected carbon atom position along the sn-2 chain (n-PCSL) were considered. On increasing the content of the lysolipids incorporated in DPPC, the lipid bilayers are first transformed in interdigitated lamellae and then converted in micelles of Lyso-PPC. In the interdigitated phase, the profile of translamellae water accessibility is rather uniform as all the hydrocarbon segments are equally exposed to the solvent. In Lyso-PPC micelle, water penetrates at any depth of the hydrocarbon region with a tendency to increase toward the chain termini. The extent of water penetration is higher in the interdigitated DPPC/Lyso-PPC dispersions than in Lyso-PPC micelles. The profiles of water permeation revealed directly by D2O-ESEEM are also confirmed by more indirect evaluation of the polarity profiles based on the 14N-hyperfine splitting in the conventional electron paramagnetic resonance spectra of n-PCSL in frozen DPPC/Lyso-PPC mixtures at 77 K. The ESEEM data reveal that H-bonding formation between the -NO group of the spin-label and the D2O molecules is favored in the intergitated phase with respect to the micellar phase and, in any lipid dispersion, the fraction of nitroxides that are singly H-bonded to deuterons is higher than the fraction that are doubly H-bonded. The overall results highlight the differences in the accessibility and properties of the solvent in the hydrocarbon region of lipid bilayers, interdigitated bilayers and micelles.

Peptide antibiotic trichogin in model membranes: Self-association and capture of fatty acids.

The antimicrobial action of peptides in bacterial membranes is commonly related to their mode of self-assembling which results in pore formation. To optimize peptide antibiotic use for therapeutic purposes, a study on the concentration dependence of self-assembling process is thus desirable. In this work, we investigate this dependence for peptaibol trichogin GA IV (Tric) in the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) model membrane in the range of peptide concentrations between 0.5 and 3.3 mol%. Pulsed double electron-electron resonance (PELDOR) applied on spin-labeled peptide analogs highlights the onset of peptide dimerization above a critical peptide concentration value, namely ~ 2 mol%. Electron spin echo (ESE) envelope modulation (ESEEM) for D2O-hydrated bilayers shows that dimerization is accompanied by peptide re-orientation towards a trans-membrane disposition. For spin-labeled stearic acids (5-DSA) in POPC bilayers, the study of ESE decays and ESEEM in the presence of a deuterated peptide analog indicates that above the critical peptide concentration the 5-DSA molecules are attracted by peptide molecules, forming nanoclusters. As the 5-DSA molecules represent a model for the behavior of fatty acids participating in bacterial membrane homeostasis, such capturing action by Tric may represent an additional mechanism of its antibiotic activity.

Feedback control optimisation of ESR experiments.

Numerically optimised microwave pulses are used to increase excitation efficiency and modulation depth in electron spin resonance experiments performed on a spectrometer equipped with an arbitrary waveform generator. The optimisation procedure is sample-specific and reminiscent of the magnet shimming process used in the early days of nuclear magnetic resonance - an objective function (for example, echo integral in a spin echo experiment) is defined and optimised numerically as a function of the pulse waveform vector using noise-resilient gradient-free methods. We found that the resulting shaped microwave pulses achieve higher excitation bandwidth and better echo modulation depth than the pulse shapes used as the initial guess. Although the method is theoretically less sophisticated than simulation based quantum optimal control techniques, it has the advantage of being free of the linear response approximation; rapid electron spin relaxation also means that the optimisation takes only a few seconds. This makes the procedure fast, convenient, and easy to use. An important application of this method is at the final stage of the implementation of theoretically designed pulse shapes: compensation of pulse distortions introduced by the instrument. The performance is illustrated using spin echo and out-of-phase electron spin echo envelope modulation experiments. Interface code between Bruker SpinJet arbitrary waveform generator and Matlab is included in versions 2.2 and later of the Spinach library.