Applications Of EPR Research Articles

Microwave field mapping for EPR-on-a-chip experiments.

Electron paramagnetic resonance-on-a-chip (EPRoC) devices use small voltage-controlled oscillators (VCOs) for both the excitation and detection of the EPR signal, allowing access to unique sample environments by lifting the restrictions imposed by resonator-based EPR techniques. EPRoC devices have been successfully used at multiple frequencies (7 to 360 gigahertz) and have demonstrated their utility in producing high-resolution spectra in a variety of spin centers. To enable quantitative measurements using EPRoC devices, the spatial distribution of the B1 field produced by the VCOs must be known. As an example, the field distribution of a 12-coil VCO array EPRoC operating at 14 gigahertz is described in this study. The frequency modulation-recorded EPR spectra of a "point"-like and a thin-film sample were investigated while varying the position of both samples in three directions. The results were compared to COMSOL simulations of the B1-field intensity. The EPRoC array sensitive volume was determined to be ~19 nanoliters. Implications for possible EPR applications are discussed.

Stabilizing Nitroxide Spin Labels for Structural and Conformational Studies of Biomolecules by Maleimide Treatment.

Nitroxide (NO) spin radicals are effective in characterizing structures, interactions and dynamics of biomolecules. The EPR applications in cell lysates or intracellular milieu require stable spin labels, but NO radicals are unstable in such conditions. We showed that the destabilization of NO radicals in cell lysates or even in cells is caused by NADPH/NADH related enzymes, but not by the commonly believed reducing reagents such as GSH. Maleimide stabilizes the NO radicals in the cell lysates by consumption of the NADPH/NADH that are essential for the enzymes involved in destabilizing NO radicals, instead of serving as the thiol scavenger. The maleimide treatment retains the crowding properties of the intracellular components and allows to perform long-time EPR measurements of NO labeled biomolecules close to the intracellular conditions. The strategy of maleimide treatment on cell lysates for the EPR applications has been demonstrated on double electron-electron resonance (DEER) measurements on a number of NO labeled protein samples. The method opens a broad application range for the NO labeled biomolecules by EPR in conditions that resemble the intracellular milieu.

A novel method for the sterically shielded pyrrolidine nitroxides synthesis using donor–acceptor cyclopropanes

Sterically shielded nitroxides were shown to demonstrate resistance to bioreduction. For this reason, they are attracting much interest as spin labels and probes for in-cell and in vivo EPR and NMR applications. Formal [3 + 2] cycloaddition reaction of donor–acceptor cyclopropanes with propionitrile provided 1-pyrrolines which were converted to the new sterically shielded achiral pyrrolidine nitroxides via several steps.

A primer in pulse EPR-based hyperfine spectroscopy for NMR spectroscopists

The theoretical foundations of NMR and EPR are very similar. Nevertheless, the fields differ significantly in practical aspects. This primer is an attempt to bridge some of the gaps between the fields, and at the same time it highlights some applications of pulse EPR. Basic EPR pulse sequences to detect nuclei in the vicinity of unpaired electrons are introduced. An emphasis is placed on explaining the concept to researchers familiar with NMR. The theoretical background as well as examples are given for electron-nuclear double resonance (ENDOR), electron-spin echo envelope modulation (ESEEM), including hyperfine sublevel correlation spectroscopy (HYSCORE), and electron double resonance (ELDOR)-detected NMR.

Revealing light-induced structural shifts in G-quadruplex-porphyrin complexes: a pulsed dipolar EPR study.

The binding of G-quadruplex structures (G4s) with photosensitizers is of considerable importance in medicinal chemistry and drug discovery due to their promising potential in photodynamic therapy applications. G4s can experience structural changes as a result of ligand interactions and light exposure. Understanding these modifications is essential to uncover the fundamental biological roles of the complexes and optimize their therapeutic potential. The structural diversity of G4s makes it challenging to study their complexes with ligands, necessitating the use of various complementary methods to fully understand these interactions. In this study, we introduce, for the first time, the application of laser-induced dipolar EPR as a method to characterize G-quadruplex DNA complexes containing photosensitizers and to investigate light-induced structural modifications in these systems. To demonstrate the feasibility of this approach, we studied complexes of the human telomeric G-quadruplex (HTel-22) with cationic 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphyrin tetra(p-toluenesulfonate) (TMPyP4). In addition to showcasing a new methodology, we also aimed to provide insights into the mechanisms underlying photoinduced HTel-22/TMPyP4 structural changes, thereby aiding in the advancement of approaches targeting G4s in photodynamic therapy. EPR revealed G-quadruplex unfolding and dimer formation upon light exposure. Our findings demonstrate the potential of EPR spectroscopy for examining G4 complexes with photosensitizers and contribute to a better understanding of G4s' interactions with ligands under light.

Towards single-cell pulsed EPR using VCO-based EPR-on-a-chip detectors

AbstractElectron paramagnetic resonance (EPR) is the gold standard for studying paramagnetic species. As an example, in structural biology, it allows to extract information about distance distributions on the nanometer scale via site-directed spin labeling. Conventional pulsed EPR of biological samples is currently limited to relatively large sample concentrations and cryogenic temperatures, mainly due to low sensitivity and the significant dead time associated with conventional resonator-based EPR setups, essentially precluding in-cell EPR under physiological conditions. This paper presents our latest progress toward single-cell pulsed EPR using VCO-based EPR-on-a-chip (EPRoC) sensors. Together with an analytical model for VCO-based pulsed EPR, we present an experimental scheme to perform dead-time-free pulsed EPR measurements using EPRoC detectors. The proposed scheme is validated using extensive numerical simulations and proof-of-concept experiments on the spin dynamics of an organic radical at room temperature using a custom-designed EPRoC detector operating in the Ka-band around 30.4 GHz. Additionally, we discuss methods to improve the excitation field homogeneity and sample handling through chip post-processing and custom-designed microfluidics. Finally, we present our progress towards compact, portable pulsed EPR spectrometers incorporating EPRoC detectors, microfluidics, and custom-designed permanent magnets. Such portable EPR spectrometers can pave the way toward new EPR applications, including point-of-care diagnostics.

Application of EPR and related methods to molybdenum-containing enzymes.

A description is provided of the contributions made to our understanding of molybdenum-containing enzymes through the application of electron paramagnetic resonance spectroscopy and related methods, by way of illustrating how these can be applied to better understand enzyme structure and function. An emphasis is placed on the use of EPR to identify both the coordination environment of the molybdenum coordination sphere as well as the structures of paramagnetic intermediates observed transiently in the course of reaction that have led to the elucidation of reaction mechanism.

RF/Microwave Resonators for Preclinical and Clinical EPR Applications: Current Status and Challenges

RF/Microwave Resonators for Preclinical and Clinical EPR Applications: Current Status and Challenges

A Review of Low-Frequency EPR Technology for the Measurement of Brain pO2 and Oxidative Stress.

EPR can uniquely measure paramagnetic species. Although commercial EPR was introduced in 1950s, the early studies were mostly restricted to chemicals in solution or cellular experiments using X-band EPR equipment. Due to its limited penetration (<1 mm), experiments with living animals were almost impossible. To overcome these difficulties, Swartz group, along with several other leaders in field, pioneered the technology of low frequency EPR (e.g., L-band, 1–2 GHz). The development of low frequency EPR and the associated probes have dramatically expanded the application of EPR technology into the biomedical research field, providing answers to important scientific questions by measuring specific parameters that are impossible or very difficult to obtain by other approaches. In this review, which is aimed at highlighting the seminal contribution from Swartz group over the last several decades, we will focus on the development of EPR technology that was designed to deal with the potential challenges arising from conducting EPR spectroscopy in living animals. The second half of the review will be concentrated on the application of low frequency EPR in measuring cerebral tissue pO2 changes and oxidative stress in various physiological and pathophysiological conditions in the brain of animal disease models.

Application of EPR to porphyrin-protein agents for photodynamic therapy

Recently, a new type of spin labels based on photoexcited triplet molecules was proposed for nanometer scale distance measurements by pulsed dipolar electron paramagnetic resonance (PD EPR). However, such molecules are also actively used within biological complexes as photosensitizers for photodynamic therapy (PDT) of cancer. Up to date, the idea of using the photoexcited triplets simultaneously as PDT agents and as spin labels for PD EPR has never been employed. In this work, we demonstrate that PD EPR in conjunction with other methods provides valuable information on the structure and function of PDT candidate complexes, exemplified here with porphyrins bound to human serum albumin (HSA). Two distinct porphyrins with different properties were used: amphiphilic meso-tetrakis(4-hydroxyphenyl)porphyrin (mTHPP) and water soluble cationic meso-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4); HSA was singly nitroxide-labeled to provide a second tag for PD EPR measurements. We found that TMPyP4 locates in a cavity at the center of the four-helix bundle of HSA subdomain IB, close to the interface with solvent, thus being readily accessible to oxygen. As a result, the photolysis of the complex leads to photooxidation of HSA by generated singlet oxygen and causes structural perturbation of the protein. Contrary, in case of mTHPP porphyrin, the binding occurs at the proton-rich pocket of HSA subdomain IIIA, where the access of oxygen to a photosensitizer is hindered. Structural data of PD EPR were supported by other EPR techniques, laser flash photolysis and protein photocleavage studies. Therefore, pulsed EPR on complexes of proteins with photoexcited triplets is a promising approach for gaining structural and functional insights into such PDT agents.

Rapid-scan EPR imaging of a phantom comprised of species with different linewidths and relaxation times.

As a demonstration of the application of rapid-scan EPR to imaging at low frequency and magnetic field, a multi-compartment phantom containing six different samples was imaged. The samples were nitroxide radicals, trityl (substituted triarylmethyl) radicals, and the oxygen-sensitive solid lithium phthalocyanine (LiPc), all of which are useful for in vivo imaging. The 2D spectral-spatial image demonstration was performed at 250 MHz, with samples in sealed tubes of various sizes arranged in a 3D-printed plastic holder. Maximum gradients of 10 G/cm gave a spatial resolution of about 0.1 mm for the narrow trityl and LiPc signals and about 1 mm for the nitroxide. The importance of proper selection of resonator bandwidth and scan rate for obtaining accurate linewidth information is demonstrated for a case in which the phantom is composed of species with signal linewidths and relaxation times that differ by more than a factor of 10.

Non-uniform HYSCORE: Measurement, processing and analysis with Hyscorean.

Non-uniform sampling (NUS) provides a considerable reduction of measurement time especially for multi-dimensional experiments. This comes at the cost of additional signal processing steps to reconstruct the complete signal from the experimental data points. Despite being routinely employed in NMR for many experiments, EPR applications have not benefited from NUS due to the lack of a straightforward implementation to perform NUS in common commercial spectrometers. In this work we present a novel method to perform NUS HYSCORE experiments on commercial Bruker EPR spectrometers, along with a benchmark of modern reconstruction methods, and new processing software tools for NUS HYSCORE signals. All of this comes in the form of a free-software package: Hyscorean. Experimental NUS spectra are measured and processed with this package using different reconstruction methods and compared to their uniform sampled counterparts, thereby showcasing the method's potential for EPR spectroscopy.

Sub‐Micromolar Pulse Dipolar EPR Spectroscopy Reveals Increasing CuII‐labelling of Double‐Histidine Motifs with Lower Temperature

AbstractElectron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to the studies of biomolecules by providing highly accurate geometric constraints. Combining double‐histidine motifs with CuII spin labels can further increase the precision of distance measurements. It is also useful for proteins containing essential cysteines that can interfere with thiol‐specific labelling. However, the non‐covalent CuII coordination approach is vulnerable to low binding‐affinity. Herein, dissociation constants (KD) are investigated directly from the modulation depths of relaxation‐induced dipolar modulation enhancement (RIDME) EPR experiments. This reveals low‐ to sub‐μm CuII KDs under EPR distance measurement conditions at cryogenic temperatures. We show the feasibility of exploiting the double‐histidine motif for EPR applications even at sub‐μm protein concentrations in orthogonally labelled CuII–nitroxide systems using a commercial Q‐band EPR instrument.

Pulse EPR in biological systems - Beyond the expert's courtyard.

Application of EPR to biological systems includes many techniques and applications. In this short perspective, which dares to look into the future, I focus on pulse EPR, which is my field of expertise. Generally, pulse EPR techniques can be divided into two main groups: (1) hyperfine spectroscopy, which explores electron-nuclear interactions, and (2) pulse-dipolar (PD) EPR spectroscopy, which is based on electron-electron spin interactions. Here I focus on PD-EPR because it has a better chance of becoming a widely applied, easy-to-use table-top method to study the structural and dynamic aspects of bio-molecules. I will briefly introduce this technique, its current state of the art, the challenges it is facing, and finally I will describe futuristic scenarios of low-cost PD-EPR approaches that can cross the diffusion barrier from the core of experts to the bulk of the scientific community.

Synthesis of hydroxyethyl tetrathiatriarylmethyl radicals OX063 and OX071.

We report the synthesis of hydroxyethyl tetrathiatriarylmethyl radical OX063 and its deuterated analogue OX071 for biomedical EPR applications.

Using optimal control methods with constraints to generate singlet states in NMR

A method is proposed for optimizing the performance of the APSOC (Adiabatic-Passage Spin Order Conversion) technique, which can be exploited in NMR experiments with singlet spin states. In this technique magnetization-to-singlet conversion (and singlet-to-magnetization conversion) is performed by using adiabatically ramped RF-fields. Optimization utilizes the GRAPE (Gradient Ascent Pulse Engineering) approach, in which for a fixed search area we assume monotonicity to the envelope of the RF-field. Such an approach allows one to achieve much better performance for APSOC; consequently, the efficiency of magnetization-to-singlet conversion is greatly improved as compared to simple model RF-ramps, e.g., linear ramps. We also demonstrate that the optimization method is reasonably robust to possible inaccuracies in determining NMR parameters of the spin system under study and also in setting the RF-field parameters. The present approach can be exploited in other NMR and EPR applications using adiabatic switching of spin Hamiltonians.

Q-Band Loop-Gap Resonator for EPR Applications with Broadband-Shaped Pulses

Recently, Q-band-pulsed electron paramagnetic resonance spectroscopy has strongly advanced its performance by the introduction of high-power microwave amplifiers and the use of shaped pulses. For such applications, the resonator Q value has to be low enough to achieve sufficient bandwidth for short microwave pulses and to reduce the ring-down time after the pulses. However, a low Q value reduces the detection sensitivity as well as the conversion efficiency of the microwave input power to the magnetic field strength at the sample position. Therefore, the resonator Q value has to be optimized for a given microwave input power and specific application. We designed a three-loop/two-gap resonator using CST Microwave Studio for such applications, and tested its performance in comparison with a standard Bruker D2 Q-band microwave resonator by accomplishing broadband SIFTER experiments on a nitroxide model compound.

Membrane remodeling by amyloidogenic and non-amyloidogenic proteins studied by EPR

The advancement in site-directed spin labeling of proteins has enabled EPR studies to expand into newer research areas within the umbrella of protein-membrane interactions. Recently, membrane remodeling by amyloidogenic and non-amyloidogenic proteins has gained a substantial interest in relation to driving and controlling vital cellular processes such as endocytosis, exocytosis, shaping of organelles like endoplasmic reticulum, Golgi and mitochondria, intracellular vesicular trafficking, formation of filopedia and multivesicular bodies, mitochondrial fusion and fission, and synaptic vesicle fusion and recycling in neurotransmission. Misregulation in any of these processes due to an aberrant protein (mutation or misfolding) or alteration of lipid metabolism can be detrimental to the cell and cause disease. Dissection of the structural basis of membrane remodeling by proteins is thus quite necessary for an understanding of the underlying mechanisms, but it remains a formidable task due to the difficulties of various common biophysical tools in monitoring the dynamic process of membrane binding and bending by proteins. This is largely since membranes generally complicate protein structure analysis and this problem is amplified for structural analysis in the presence of different types of membrane curvatures. Recent EPR studies on membrane remodeling by proteins show that a significant structural information can be generated to delineate the role of different protein modules, domains and individual amino acids in the generation of membrane curvature. These studies also show how EPR can complement the data obtained by high resolution techniques such as X-ray and NMR. This perspective covers the application of EPR in recent studies for understanding membrane remodeling by amyloidogenic and non-amyloidogenic proteins that is useful for researchers interested in using or complimenting EPR to gain better understanding of membrane remodeling. We also discuss how a single protein can generate different type of membrane curvatures using specific conformations for specific membrane structures and how EPR is a versatile tool well-suited to analyze subtle alterations in structures under such modifying conditions which otherwise would have been difficult using other biophysical tools.

Applications of EPR and NMR Spectroscopy in Homogeneous Catalysis

Applications of EPR and NMR Spectroscopy in Homogeneous Catalysis

Nanoscale lipid membrane mimetics in spin-labeling and electron paramagnetic resonance spectroscopy studies of protein structure and function

AbstractCellular membranes and associated proteins play critical physiological roles in organisms from all life kingdoms. In many cases, malfunction of biological membranes triggered by changes in the lipid bilayer properties or membrane protein functional abnormalities lead to severe diseases. To understand in detail the processes that govern the life of cells and to control diseases, one of the major tasks in biological sciences is to learn how the membrane proteins function. To do so, a variety of biochemical and biophysical approaches have been used in molecular studies of membrane protein structure and function on the nanoscale. This review focuses on electron paramagnetic resonance with site-directed nitroxide spin-labeling (SDSL EPR), which is a rapidly expanding and powerful technique reporting on the local protein/spin-label dynamics and on large functionally important structural rearrangements. On the other hand, adequate to nanoscale study membrane mimetics have been developed and used in conjunction with SDSL EPR. Primarily, these mimetics include various liposomes, bicelles, and nanodiscs. This review provides a basic description of the EPR methods, continuous-wave and pulse, applied to spin-labeled proteins, and highlights several representative applications of EPR to liposome-, bicelle-, or nanodisc-reconstituted membrane proteins.