Spin Probe Research Articles

Computation of Overhauser dynamic nuclear polarization processes reveals fundamental correlation between water dynamics, structure, and solvent restructuring entropy.

Hydration water dynamics, structure, and thermodynamics are crucially important to understand and predict water-mediated properties at molecular interfaces. Yet experimentally and directly quantifying water behavior locally near interfaces at the sub-nanometer scale is challenging, especially at interfaces submerged in biological solutions. Overhauser dynamic nuclear polarization (ODNP) experiments measure equilibrium hydration water dynamics within 8-15 angstroms of a nitroxide spin probe on instantaneous timescales (10 picoseconds to nanoseconds), making ODNP a powerful tool for probing local water dynamics in the vicinity of the spin probe. As with other spectroscopic techniques, concurrent computational analysis is necessary to gain access to detailed molecular level information about the dynamic, structural, and thermodynamic properties of water from experimental ODNP data. We chose a model system that can systematically tune the dynamics of water, a water-glycerol mixture with compositions ranging from 0 to 0.3 mole fraction glycerol. We demonstrate the ability of molecular dynamics (MD) simulations to compute ODNP spectroscopic quantities, and show that translational, rotational, and hydrogen bonding dynamics of hydration water align strongly with spectroscopic ODNP parameters. Moreover, MD simulations show tight correlations between the dynamic properties of water that ODNP captures and the structural and thermodynamic behavior of water. Hence, experimental ODNP readouts of varying water dynamics suggest changes in local structural and thermodynamic hydration water properties.

Singlet Oxygen Detection by Chemiluminescence Probes in Living Cells.

Singlet oxygen is a reactive oxygen species that causes oxidative damage to plant cells, but intriguingly it can also act as a signalling molecule to reprogram gene expression required to induce plant physiological/cellular responses. Singlet oxygen photosensitization in plants mainly occurs in chloroplasts after the molecular collision of ground-state molecular oxygen with triplet-excited-state chlorophyll. Singlet oxygen direct detection through phosphorescence emission in chloroplasts is a herculean task due to its extremely low luminescence quantum yield. Because of this, indirect alternative methods have been developed for its detection in biological systems, for example, by measuring the changes in the EPR signal or fluorescence intensity of singlet oxygen reaction-based probes. The singlet oxygen chemiluminescence (SOCL) is a chemiluminescence probe with high sensitivity and selectivity towards singlet oxygen and promising use to detect it in living cells without the inconvenience of low stability of the EPR signal of spin probes in the presence of redox compounds, spurious light scattering coming from the light source required for the excitation of fluorescence probes or the light emission of endogenous fluorescent molecules like chlorophyll in chloroplasts. The protocol presented in this chapter describes the first steps to characterizing singlet oxygen production within the biological system under study; this is accomplished through monitoring molecular oxygen consumption by SOCL using a Clark-type oxygen electrode and measuring the chemiluminescence generated by SOCL 1,2-dioxetane using a spectrofluorometer. For singlet oxygen detection within living cells, a version of SOCL with increased membrane permeability (SOCL-CPP) is described.

Understanding the effects of targeted modifications on the 1 : 2 Choline And GEranate structure.

1 : 2 Choline-and-geranate (CAGE) is an ionic liquid (IL) widely studied for its biomedical applications. However, both its industrial-scale preparation and its long-term storage are problematic so finding more suitable candidates which retain its advantageous properties is crucial. As a first step towards this we have conducted a targeted modification study to understand the effects of specific functional groups on the properties of CAGE. 1 : 2 Choline-and-octanoate and 1 : 2 butyltrimethylammonium-and-octanoate were synthesised and their thermal and rheological properties examined in comparison to those of CAGE. Using differential scanning calorimetry and polarising microscopy, the model compound was found to be an isotropic liquid, while the analogues were room-temperature liquid-crystals which transition to isotropic liquids upon heating. Dynamic mechanical analysis showed that the thermal behaviour of the studied systems was even more complex, with the ILs also undergoing a thermally-activated relaxation process. Furthermore, we have used electron paramagnetic resonance (EPR) spectroscopy, along with a variety of spin probes with different functional groups, in order to understand the chemical environment experienced by solutes in each system. The EPR spectra indicate that the radicals experience two distinct environments (polar and nonpolar) in the liquid-crystalline phase, but only one average environment in the isotropic phase. The liquid-crystalline phase experiments also showed that the relative populations of the two domains depend on the nature of the solutes, with polar or strongly hydrogen-bonding solutes preferring the polar domain. For charged solutes, the EPR spectra showed line-broadening, suggesting that their ionic nature leads to complex, unresolved interactions.

Quantitative Ensemble Interpretation of Membrane Paramagnetic Relaxation Enhancement (mPRE) for Studying Membrane-Associated Intrinsically Disordered Proteins.

An understanding of the functional role played by a membrane-associated intrinsically disordered protein (IDP) requires characterization of its heterogeneous conformations as well as its poses relative to the membranes, which is of great interest but technically challenging. Here, we explore the membrane paramagnetic relaxation enhancement (mPRE) for constructing ensembles of IDPs that dynamically associate with membrane mimetics incorporating spin-labeled lipids. To accurately interpret the mPRE Γ2 rates, both the dynamics of IDPs and spin probe molecules are taken into account, with the latter described by a weighted three-dimensional (3D) grid model built based on all-atom simulations. The IDP internal conformations, orientations, and immersion depths in lipid bilayers are comprehensively optimized in the Γ2-based ensemble modeling. Our approach is tested and validated on the example of POPG bicelle-bound disordered cytoplasmic domain of CD3ε (CD3εCD), a component of the T-cell receptor (TCR) complex. The mPRE-derived CD3εCD ensemble provides new insights into the IDP-membrane fuzzy association, in particular for the tyrosine-based signaling motif that plays a critical role in TCR signaling. The comparative analysis of the ensembles for wild-type CD3εCD and mutants that mimic the mono- and dual-phosphorylation effects suggests a delicate membrane regulatory mechanism for activation and inhibition of the TCR activity.

Diffusion of the Tempone Spin Probe in Poly(Methyl Methacrylate) and Poly(D,L-Lactide) Plasticized with Sub- and Supercritical CO2

Diffusion of the Tempone Spin Probe in Poly(Methyl Methacrylate) and Poly(D,L-Lactide) Plasticized with Sub- and Supercritical CO2

Electron Spin Resonance Probe Incorporation into Bioinks Permits Longitudinal Oxygen Imaging of Bioprinted Constructs

PurposeBioprinting is an additive manufacturing technology analogous to 3D printing. Instead of plastic or resin, cell-laden hydrogels are used to produce a construct of the intended biological structure. Over time, cells transform this construct into a functioning tissue or organ. The process of printing followed by tissue maturation is referred to as 4D bioprinting. The fourth dimension is temporal. Failure to provide living cells with sufficient amounts of oxygen at any point along the developmental timeline may jeopardize the bioprinting goals. Even transient hypoxia may alter cells' differentiation and proliferation or trigger apoptosis. Electron paramagnetic resonance (EPR) imaging modality is proposed to permit 4D monitoring of oxygen within bioprinted structures.ProceduresLithium octa-n-butoxy-phthalocyanine (LiNc-BuO) probes have been introduced into gelatin methacrylate (GelMA) bioink. GelMA is a cross-linkable hydrogel, and LiNc-BuO is an oxygen-sensitive compound that permits longitudinal oximetric measurements. The effects of the oxygen probe on printability have been evaluated. A digital light processing (DLP) bioprinter was built in the laboratory. Bioprinting protocols have been developed that consider the optical properties of the GelMA/LiNc-BuO composites. Acellular and cell-laden constructs have been printed and imaged. The post-printing effect of residual photoinitiator on oxygen depletion has been investigated.ResultsModels have been successfully printed using a lab-built bioprinter. Rapid scan EPR images reflective of the expected oxygen concentration levels have been acquired. An unreported problem of oxygen depletion in bioprinted constructs by the residual photoinitiator has been documented. EPR imaging is proposed as a control method for its removal. The oxygen consumption rates by HEK293T cells within a bioprinted cylinder have been imaged and quantified.ConclusionsThe feasibility of the cointegration of 4D EPR imaging and 4D bioprinting has been demonstrated. The proof-of-concept experiments, which were conducted using oxygen probes loaded into GelMA, lay the foundation for a broad range of applications, such as bioprinting with many types of bioinks loaded with diverse varieties of molecular spin probes.

Gadolinium Spin Decoherence Mechanisms at High Magnetic Fields.

Favorable relaxation processes, high-field spectral properties, and biological compatibility have made spin-7/2 Gd3+-based spin labels an increasingly popular choice for protein structure studies using high-field electron paramagnetic resonance. However, high-field relaxation and decoherence in ensembles of half-integer high-spin systems, such as Gd3+, remain poorly understood. We report spin-lattice (T1) and phase memory (TM) relaxation times at 8.6 T (240 GHz), and we present the first comprehensive model of high-field, high-spin decoherence accounting for both the electron spin concentration and temperature. The model includes four principal mechanisms driving decoherence: energy-conserving electron spin flip-flops, direct "T1" spin-lattice relaxation-driven electron spin flip processes, indirect T1-driven flips of nearby electron spins, and nuclear spin flip-flops. Mechanistic insight into decoherence can inform the design of experiments making use of Gd3+ as spin probes or relaxivity agents and can be used to measure local average interspin distances as long as 17 nm.

A New Quantitative Method of Express Analysis of Natural Rubber Directly in the Root of the Rubber Plant by the Spin Probe EPR Method

A New Quantitative Method of Express Analysis of Natural Rubber Directly in the Root of the Rubber Plant by the Spin Probe EPR Method

Study of factors affecting some properties of hydrophilic suppository base

The aim. To study the effect of the composition of hydrophilic suppository bases on the physicochemical and osmotic properties of suppositories made from them.
 Materials and methods. The bases were studied with varying compositions of excipients. The microstructure of the bases was evaluated, and the disintegration time and resistance to rupture of suppositories made from them were determined. The kinetics of water absorption and solvent release were studied by dialysis. The content of propylene glycol (PG) and macrogol 400 (M400) in the receptor medium was determined by gas chromatography. The melted bases were studied by rotational viscometry. The electron paramagnetic resonance spectra of spin probes in hydrophilic solvents and bases were obtained; the type of spectrum, isotropic constant (AN), rotational correlation times (τ), and anisotropy parameter (ε) were determined.
 Results. The disintegration times and resistance to rupture of suppositories were determined depending on such factors as the content and grade of poloxamers, the ratio between high molecular weight macrogols and the mixed solvent PG-M400 (60 : 40 % m/m), the ratio of nonionic surfactant and cetostearyl alcohol (CSA) and their total content, water and hard fat content. The introduction of solid fat and a mixture of surfactants and CSA provides the uniform structure of the bases. The mass ratio between surfactants and CSA and their total content are important factors that provide acceptable resistance to rupture and disintegration times for suppositories and reduce water absorption and solvent release. As the temperature decreases from 45 °C to 20 °C, the bases transform from Newtonian liquids to solids. At that time, the microviscosity of the environment of the spin probes increased by about 5 times, but the parameters of their rotational diffusion in solid bases and the mixed solvent PG-M400 are comparable. This indicates the dissolved state of the spin probes in the bases and the absence of the formation of mixed associates from molecules of surfactant and CSA.
 Conclusions. By varying the composition of excipients, the properties of hydrophilic suppository bases can be controlled, significantly reducing their osmotic properties. The active substances in these bases may be in a dissolved state due to the high content of non-aqueous solvents

Synergistic removal of organic and inorganic composite pollutants from water by zero-valent iron activated calcium peroxide

Agricultural non-point source pollution has received widespread attention in recent years due to its characteristics of dispersion, uncertainty, extensiveness and high difficulty in control. Among them, components such as fertilizers, heavy metals and herbicides constitute complex inorganic–organic composite pollution, which poses great threat to agricultural water ecosystem. Therefore, in this study, phosphate (P), lead (Pb) and atrazine (ATZ) as typical pollutants from agricultural non-point sources were investigated to remove collaboratively by constructing an environmentally friendly zero-valent iron (ZVI)-activated calcium peroxide (CaO2) system. At unadjusted initial pH 2.86, with the mass ratio of ZVI/CaO2 1:1, the removal efficiencies of P, Pb and ATZ were 95.3%, 97.4%, and virtually 100%, respectively. The main active species in the ZVI/CaO2 system was identified as hydroxyl radicals (·OH) by quenching experiment, electron spin resonance test and probe experiment. The characterization results showed that P was mainly precipitated in the form of Ca-P and Fe-P species. Pb was mainly adsorbed and reduced by ZVI and precipitated in solution. ATZ was mainly degraded into ten by-products, and the overall toxicity showed a decrease. The influence of pH on the removal efficiencies of pollutants was reflected in the fact that high pH promoted the removal of P and Pb but inhibited the degradation of ATZ. The ZVI/CaO2 system performed well under the influence of humic acid and typical anions. Therefore, this work provides a promising technology for the organic–inorganic co-polluted water treatment.

Oligomeric and Fibrillar α-Synuclein Display Persistent Dynamics and Compressibility under Controlled Confinement.

The roles of α-synuclein in neurotransmitter release in brain neurons and in the Parkinson's disease condition have challenged comprehensive description. To gain insight into molecular mechanistic properties that actuate α-synuclein function and dysfunction, the coupled protein and solvent dynamics of oligomer and fibril forms of human α-synuclein are examined in a low-temperature system that allows control of confinement and localization of a motionally sensitive electron paramagnetic resonance spin probe in the coupled solvent-protein regions. The rotational mobility of the spin probe resolves two distinct α-synuclein-associated solvent components for oligomers and fibrils, as for globular proteins, but with dramatically higher fluidities at each temperature, that are comparable to low-confinement, aqueous-cryosolvent mesophases. In contrast to the temperature-independent volumes of the solvent phases that surround globular and condensate-forming proteins, the higher-fluidity mesophase volume of α-synuclein oligomers and fibrils decreases with decreasing temperature, signaling a compression of this phase. This unique property and thermal hysteresis in the mobilities and component weights, together with previous high-resolution structural characterizations, suggest a model in which the dynamically disordered C-terminal domain of α-synuclein creates a compressible phase that maintains high fluidity under confinement. Robust dynamics and compressibility are fundamental molecular mechanical properties of α-synuclein oligomers and fibrils, which may contribute to dysfunction and inform about function.

Heisenberg Spin Exchange Between Nitroxide Probes Diffusing in a Percolation Network

AbstractHeisenberg spin exchange between nitroxide (Tempone) spin probes has been measured as a function of concentration in the aqueous phase of the hydrated ion exchange membrane Nafion 117. The observed fast-motional electron paramagnetic resonance spectra were analyzed in terms of the stochastic Liouville equation lineshape calculation of Freed and coworkers and the “new paradigm” for interpreting spin exchange effects proposed by Salikhov. Differences between the effective spin exchange measured from the spectrum by these methods are presented and compared, and indicate that dipolar interactions make a significant contribution to spin exchange in this system. In acidic Nafion membranes, the spin probes are deactivated over time, allowing simultaneous measurement of the decay kinetics and spin exchange as a function of paramagnetic probe concentration. Both these processes deviate from the behavior that would be expected from classical chemical kinetics in isotropic media. The results are discussed in terms of currently available models for diffusion and reaction in a percolation network.

Pulse and CW EPR Oximetry Using Oxychip in Gemcitabine-Treated Murine Pancreatic Tumors

PurposeThe goal of this work was to compare pO2 measured using both continuous wave (CW) and pulse electron paramagnetic resonance (EPR) spectroscopy. The Oxychip particle spin probe enabled longitudinal monitoring of pO2 in murine pancreatic tumor treated with gemcitabine during the course of therapy.ProceduresPancreatic PanO2 tumors were growing in the syngeneic mice, in the leg. Five doses of saline in control animals or gemcitabine were administered every 3 days, and pO2 was measured after each dose at several time points. Oxygen partial pressure was determined from the linewidth of the CW EPR signal (Bruker E540L) or from the T2 measured using the electron spin echo sequence (Jiva-25™).ResultsThe oxygen sensitivity was determined from a calibration curve as 6.1 mG/mm Hg in CW EPR and 68.5 ms−1/mm Hg in pulse EPR. A slight increase in pO2 of up to 20 mm Hg was observed after the third dose of gemcitabine compared to the control. The maximum delta pO2 during the therapy correlated with better survival.ConclusionsBoth techniques offer fast and reliable oximetry in vivo, allowing to follow the effects of pharmaceutic intervention.

In vitro oxygen imaging of acellular and cell-loaded beta cell replacement devices

Type 1 diabetes (T1D) is an autoimmune disease that leads to the loss of insulin-producing beta cells. Bioartificial pancreas (BAP) or beta cell replacement strategies have shown promise in curing T1D and providing long-term insulin independence. Hypoxia (low oxygen concentration) that may occur in the BAP devices due to cell oxygen consumption at the early stages after implantation damages the cells, in addition to imposing limitations to device dimensions when translating promising results from rodents to humans. Finding ways to provide cells with sufficient oxygenation remains the major challenge in realizing BAP devices’ full potential. Therefore, in vitro oxygen imaging assessment of BAP devices is crucial for predicting the devices’ in vivo efficiency. Electron paramagnetic resonance oxygen imaging (EPROI, also known as electron MRI or eMRI) is a unique imaging technique that delivers absolute partial pressure of oxygen (pO2) maps and has been used for cancer hypoxia research for decades. However, its applicability for assessing BAP devices has not been explored. EPROI utilizes low magnetic fields in the mT range, static gradients, and the linear relationship between the spin–lattice relaxation rate (R1) of oxygen-sensitive spin probes such as trityl OX071 and pO2 to generate oxygen maps in tissues. With the support of the Juvenile Diabetes Research Foundation (JDRF), an academic-industry partnership consortium, the “Oxygen Measurement Core” was established at O2M to perform oxygen imaging assessment of BAP devices originated from core members’ laboratories. This article aims to establish the protocols and demonstrate a few examples of in vitro oxygen imaging of BAP devices using EPROI. All pO2 measurements were performed using a recently introduced 720 MHz/25 mT preclinical oxygen imager instrument, JIVA-25™. We began by performing pO2 calibration of the biomaterials used in BAPs at 25 mT magnetic field since no such data exist. We compared the EPROI pO2 measurement with a single-point probe for a few selected materials. We also performed trityl OX071 toxicity studies with fibroblasts, as well as insulin-producing cells (beta TC6, MIN6, and human islet cells). Finally, we performed proof-of-concept in vitro pO2 imaging of five BAP devices that varied in size, shape, and biomaterials. We demonstrated that EPROI is compatible with commonly used biomaterials and that trityl OX071 is nontoxic to cells. A comparison of the EPROI with a fluorescent-based point oxygen probe in selected biomaterials showed higher accuracy of EPROI. The imaging of typically heterogenous BAP devices demonstrated the utility of obtaining oxygen maps over single-point measurements. In summary, we present EPROI as a quality control tool for developing efficient cell transplantation devices and artificial tissue grafts. Although the focus of this work is encapsulation systems for diabetes, the techniques developed in this project are easily transferable to other biomaterials, tissue grafts, and cell therapy devices used in the field of tissue engineering and regenerative medicine (TERM). In summary, EPROI is a unique noninvasive tool to experimentally study oxygen distribution in cell transplantation devices and artificial tissues, which can revolutionize the treatment of degenerative diseases like T1D.

Oxygen Imaging of a Rabbit Tumor Using a Human-Sized Pulse Electron Paramagnetic Resonance Imager.

Spatial heterogeneity in tumor hypoxia is one of the most important factors regulating tumor growth, development, aggressiveness, metastasis, and affecting treatment outcome. Most solid tumors are known to have hypoxia or low oxygen levels (pO2 ≤10 torr). Electron paramagnetic resonance oxygen imaging (EPROI) is an emerging oxygen mapping technology. EPROI utilizes the linear relationship between the relaxation rates of the injectable OX071 trityl spin probe and the partial oxygen pressure (pO2). However, most of the EPROI studies have been limited to mouse models of solid tumors because of the instrument-size limitations. The purpose of this work was to develop a human-sized 9-mT (250 MHz resonance frequency, 60 cm bore size) pulse EPROI instrument and evaluate its performance with rabbit VX-2 tumor oxygen imaging. A New Zealand white rabbit with a 3.2-cm VX-2 tumor in the calf muscle was imaged using the human-sized EPROI instrument and a 2.25-in. ID volume coil. The animal received a ~8-min intravenous injection of OX071 (5.2 mL total volume at 72 mM concentration) and, after 75 min, an intratumoral injection (120 μL total at 5 mM OX071 concentration) and underwent EPROI. At the end of the experiments, MRI was performed using a preclinical 9.4-T MRI system to outline the tumor boundaries. For the first time, a human-sized pulse EPROI instrument with a 60-cm bore size/250-MHz frequency was built and evaluated using rabbit tumor oxygen imaging. For the first time, the systemic IV injection of the oxygen-sensitive trityl OX071 spin probe was used for an animal of this size. The resulting EPROI image from the IV injection showed complete tumor coverage. The image obtained after intratumoral injection showed localized coverage in the upper lobe of the tumor, demonstrating the need for improved intratumoral injection protocol. This study demonstrates the performance of the world's first human-sized pulse EPROI instrument. It also demonstrates that the EPROI of larger animals can be performed using the systemic injection of a manageable amount of the spin probe. This brings EPROI one step closer to clinical applications in cancer therapies. Oxygen imaging is a platform technology, and the instrument and techniques developed here will also be useful for other clinical applications.

Evaluation of a deuterated triarylmethyl spin probe for in vivo R2 ∗-based EPR oximetric imaging with enhanced dynamic range.

In this study, we compared two triarylmethyl (TAM) spin probes, Ox071 and Ox063 for their efficacy in measuring tissue oxygen levels under hypoxic and normoxic conditions by R2 *-based EPR oximetry. The R2 * dependencies on the spin probe concentration and oxygen level were calibrated using deoxygenated 1, 2, 5, and 10 mM standard solutions and 2 mM solutions saturated at 0%, 2%, 5%, 10%, and 21% of oxygen. For the hypoxic model, in vivo imaging of a MIA PaCa-2 tumor implanted in the hind leg of a mouse was performed on successive days by R2 *-based EPR oximetry using either Ox071 or Ox063. For the normoxic model, renal imaging of healthy athymic mice was performed using both spin probes. The 3D images were reconstructed by single point imaging and multi-gradient technique was used to determine R2 * maps. The signal intensities of Ox071 were approximately three times greater than that of Ox063 in the entire partial pressure of oxygen (pO2 ) range investigated. The histograms of the tumor pO2 images were skewed for both spin probes, and Ox071 showed more frequency counts at pO2 > 32 mm Hg. In the normoxic kidney model, there was a clear delineation between the high pO2 cortex and the low pO2 medulla regions. The histogram of high-resolution kidney oximetry image using Ox071 was nearly symmetrical and frequency counts were seen up to 55 mm Hg, which were missed in Ox063 imaging. As an oximetric probe, Ox071 has clear advantages over Ox063 in terms of sensitivity and the pO2 dynamic range.

Study of aqueous solutions of poloxamers by rotational viscometry and spin probe method

The aim. To study aqueous solutions of different poloxamers by spin probe method and rotational viscometry depending on the temperature and poloxamer content.
 Materials and methods. The aqueous solutions of poloxamers 188, 237, 338 and 407 were studied. The solutions were studied by rotational viscometry at different temperatures; the flow behaviour, yield stress (t0), and dynamic or apparent viscosity (η) were determined. Five spin probes differing in molecular structure, solubility, and radical localisation were introduced into the solutions. Electron paramagnetic resonance (EPR) spectra were obtained. The EPR spectra were used to determine their type and to calculate parameters.
 Results. Three factors are important for gel formation: the poloxamer type, its concentration in aqueous solution, and temperature. As the temperature of aqueous solutions of poloxamers 237, 338, and 407 increases, the rotational correlation times of fatty acid-based spin probes and the order parameters of their EPR spectra decrease. This indicates a decrease in the packing density and orderliness of the polypropylene oxide (PPO) chains in the non-polar part of the poloxamer associates, leading to an increase in the volume fraction of micelles/mesophases and promoting the formation of gels. As the temperature decreases, the opposite processes occur, leading to a gel → sol transition. At 37 °C, non-polar micelle cores could be characterised as two-dimensionally liquid and one-dimensionally solid. The rotational correlation times of the hydrophilic spin probe 4-OXO-TEMPO in 25 % aqueous solutions of poloxamers 338 and 407 are approximately constant or increasing despite an increase in the temperature. This indicates that in the polar part of the poloxamer associates, where this probe is partially localised, structural rearrangements occur with increasing temperature, which probably prevents hydrophobic hydration of the PPO chains.
 Conclusions. The rheological properties of aqueous solutions of poloxamers depend on their type, concentration, and temperature. According to the parameters of the EPR spectra of fatty acid-based spin probes, it was found that with increasing temperature, the packing density and the orderliness of the PPO chains in the non-polar part of the poloxamer associates decrease, probably leading to an increase in the volume of the micelles and causing a sol → gel transition

Native and nonnative reactions in ethanolamine ammonia-lyase are actuated by different dynamics

We address the contribution of select classes of solvent-coupled configurational fluctuations to the complex choreography involved in configurational and chemical steps in an enzyme by comparing native and nonnative reactions conducted at different protein internal sites. The low temperature, first-order kinetics of covalent bond rearrangement of the cryotrapped substrate radical in coenzyme B12-dependent ethanolamine ammonia-lyase (EAL) from Salmonella enterica display a kink, or increase in slope, of the Arrhenius plot with decreasing temperature. The event is associated with quenching of a select class of reaction-actuating collective fluctuations in the protein hydration layer. For comparison, a nonnative, radical reaction of the protein interior cysteine sulfhydryl group with hydrogen peroxide (H2O2) is introduced by cryotrapping EAL in an aqueous H2O2 eutectic system. The low-temperature aqueous H2O2 protein hydration and mesodomain solvent phases surrounding cryotrapped EAL are characterized by using TEMPOL spin probe electron paramagnetic resonance spectroscopy, including a freezing transition of the eutectic phase that orders the protein hydration layer. Kinetics of the cysteine-H2O2 reaction in the EAL protein interior are monitored by DEPMPO spin trapping of hydroxyl radical product. In contrast to the native reaction, the linear Arrhenius relation for the nonnative cysteine-H2O2 reaction is maintained through the solvent-protein ordering transition. The nonnative reaction is coupled to the generic local, incremental fluctuations that are intrinsic to globular proteins. The comparative approach supports the proposal that select coupled solvent-protein configurational fluctuations actuate the native reaction, and suggests that select dynamical coupling contributes to the degree of catalysis in enzymes.

Resin–Paraffin Aggregation Processes in Waxy Oil Revealed by Vanadyl Porphyrin Spin Probe

Resin–Paraffin Aggregation Processes in Waxy Oil Revealed by Vanadyl Porphyrin Spin Probe

Pulse EPR spectroscopy and molecular modeling reveal the origins of the local heterogeneity of dietary fibers

Optimizing human diet by including dietary fibers would be more efficient when the fibers' chain interactions with other molecules are understood in depth. Thereby, it is important to develop methods for characterizing the fiber chain to be able to monitor its structural alterations upon intermolecular interactions. Here, we demonstrate the utility of the electron paramagnetic resonance (EPR) spectroscopy, complemented by simulations in probing the atomistic details of the chain conformations for spin-labeled fibers. Barley β-glucan, a native polysaccharide with linear chain, was utilized as a test fiber system to demonstrate the technique's capabilities. Pulse dipolar EPR data show good agreement with results of the fiber chain modeling, revealing sinuous chain conformations and providing polymer shape descriptors: the gyration tensor, spin-spin distance distribution function, and information about proton density near the spin probe. Results from EPR measurements point to the fiber aggregation in aqueous solution, which agrees with the results of the dynamic light scattering. We propose that the combination of pulse EPR measurements with modeling can be a perfect experimental tool for in-depth structural investigation of dietary fibers and their interaction under such conditions, and that the presented methodology can be extended to other weakly ordered or disordered macromolecules.