Spin Probe Molecules Research Articles

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.

Harnessing Electron Spin Hyperpolarization in Chromophore-Radical Spin Probes for Subcellular Resolution in Electron Paramagnetic Resonance Imaging: Concept and Feasibility.

Obtaining a subcellular resolution for biological samples doped with stable radicals at room temperature (RT) is a long-sought goal in electron paramagnetic resonance imaging (EPRI). The spatial resolution in current EPRI methods is constrained either because of low electron spin polarization at RT or the experimental limitations associated with the field gradients and the radical linewidth. Inspired by the recent demonstration of a large electron spin hyperpolarization in chromophore-nitroxyl spin probe molecules, the present work proposes a novel optically hyperpolarized EPR imaging (OH-EPRI) method, which combines the optical method of two-photon confocal microscopy for hyperpolarization generation and the rapid scan (RS) EPR method for signal detection. An important aspect of OH-EPRI is that it is not limited by the abovementioned restrictions of conventional EPRI since the large hyperpolarization in the spin probes overcomes the poor thermal spin polarization at RT, and the use of two-photon optical excitation of the chromophore naturally generates the required spatial resolution, without the need for any magnetic field gradient. Simulations based on time-dependent Bloch equations, which took into account both the RS field modulation and the hyperpolarization generation by optical means, were performed to examine the feasibility of OH-EPRI. The simulation results revealed that a spatial resolution of up to 2 fL can be achieved in OH-EPRI at RT under in vitro conditions. Notably, the majority of the requirements for an OH-EPRI experiment can be fulfilled by the currently available technologies, thereby paving the way for its easy implementation. Thus, the proposed method could potentially bridge the sensitivity gap between the optical and magnetic imaging techniques.

Impregnation of Polycarbonate by Paramagnetic Probe 2,2,6,6-Tetramethyl-4-Hydroxy-Piperidine-1-Oxyl (TEMPOL) in Supercritical CO2

The aim of the research was to test the advantages of spin probe electron paramagnetic resonance approach in studying polymers impregnation with organic molecules in supercritical CO2 (scCO2) The impregnation of bisphenol A polycarbonate with the spin probe TEMPOL was carried out at 307–343 K and 11.6–35 MPa. The mean and local concentrations of the spin probe in the polymer were evaluated. An increase in temperature and pressure resulted in a more even distribution of the dopant in the polymer matrix. It was observed that, at 307 K and 19.6 MPa, the spin probe was located only near the surface of the sample. Local mobility of the spin probe molecules was found to be similar in polycarbonate films impregnated in scCO2 and cast from dichloroethane solution. It was shown that changes in the structure of the surface and bulk of the polymer detected by the atomic force and optical polarization microscopy are not directly related with the distribution of the dopant molecules and their average content in the polymer.

Impregnation of polymers with 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TEMPONE) paramagnetic probe in sub- and supercritical CO2

The spin probe method is used to study the impregnation of polycarbonate (PC) based on bisphenol A, polyethylene oxide (PEO), and crosslinked acrylamide–acrylic acid copolymer (PAA) with organic molecules in sub- and supercritical CO2 media. Electron spin resonance (EPR) data show that, at 196 bar and 307 K, 2,2,6,6-tetramethyl-4-oxo-piperidine-1-oxyl (TEMPONE) paramagnetic spin probe molecules penetrate into the PC and PEO matrices, which are, respectively, in the glassy and elastic states under normal conditions. The degree of impregnation of PAA under these conditions is negligibly small. Estimates of the local concentration of probe molecules show that, in the PEO matrix, TEMPONE is distributed much more uniformly than in the PC matrix. Analysis of the effect of temperature on the shape of the EPR spectra of the radical in the polymer matrix shows that, under the same conditions, the mobility of TEMPONE molecules in the PEO matrix is much higher than in the PC matrix. The results suggest that the spin probe method is promising for studying the characteristics of macro- and micro-processes in polymer–supercritical fluid solvent–organic molecule ternary systems.

Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Solvation of Small Disulfonate Anions in Water/Methanol Mixtures Characterized by High-Field Pulse Electron Nuclear Double Resonance and Molecular Dynamics Simulations

The solvation of Fremy's salt, the paramagnetic nitrosodisulfonate anion ON(SO(3)(-))(2), in binary solvent mixtures was investigated by means of pulse (Mims- and Davies-type) electron nuclear double resonance (ENDOR) spectroscopy and molecular dynamics (MD) simulations. (1)H and (2)H pulse ENDOR measurements were performed on small Fremy's salt radicals in isotope-substituted solvent mixtures of methanol and water in frozen solution. We were able to obtain well-resolved, orientation-selective, high-field/high-frequency pulse ENDOR spectra of methyl protons from the alcohol moiety and exchangeable protons from the alcohol-hydroxyl group and water. In the studied solvent systems (volume ratio v/v = 30:70, 50:50, 70:30), the solvation of 2.5 mM Fremy's salt by methyl protons was found to be almost identical. From the analysis of the dependence of pulse ENDOR spectra on the observer field position and spectral simulations, we obtained the principal components of the hyperfine coupling (hfc) tensor for each class of protons. The combination of Mims- and Davies-type pulse ENDOR measurements was necessary to obtain blind spot free information on hfc that spans a broad range of 0.25-6 MHz. Using the point-dipole approximation, the dipolar hfc component yields a prominent electron-nuclear distance of 3.5 A between Fremy's salt and methyl protons, which was found along the molecular z-axis (perpendicular to the approximate plane spanned by ON(S)(2)) of the probe molecule. Exchangeable protons were found to be distributed nearly isotropically, forming a hydrogen-bonded network around the sulfonate groups. The distribution of exchangeable and methyl protons found in MD simulations is in very good agreement with the pulse ENDOR results, and we find that solvation is dominated by an interplay of H-bond (electrostatic) interactions and steric properties. The elucidation of the microscopic solvation of a small probe molecule in binary solvent mixtures represents the first step for understanding the interactions in more complex biochemical systems. In particular, this includes the potential perturbation of the H-bond network due to the presence of a spin probe or other polar molecules.

Spin-probe ESR and molecular modeling studies on calcium carbonate dispersions in overbased detergent additives

Oil-soluble calcium carbonate colloids are used as detergent additives in lubricating oils. They are colloidal dispersions of calcium carbonate particles stabilized by different surfactants; in this study alkyl-aryl-sulfonates and sulfurized alkyl-phenates, widely used in the synthesis of these additives, are considered. The physical properties of surfactant layers surrounding the surfaces of calcium carbonate particles were analyzed by using some nitroxide spin-probes (stable free radicals) and observing the corresponding ESR spectra. The spin-probe molecules contain polar groups which tend to tether them to the carbonate particle polar surface. They can reach these surfaces only if the surfactant layers are not very compact, hence the relative amounts of spin-probe molecules accessing carbonate surfaces are an index of the compactness of surfactant core. ESR signals of spin-probe molecules dissolved in oil or “locked” near the carbonate surfaces are different because of the different molecular mobility. Through deconvolution of the ESR spectra, the fraction of spin-probes penetrating surfactant shells have been calculated, and differences were observed according to the surfactant molecular structures. Moreover, by using specially labeled spin-probes based on stearic acids, functionalized at different separations from the carboxylic acid group, it was possible to interrogate the molecular physical behavior of surfactant shells at different distances from carbonate surfaces. Molecular modeling was applied to generate some three-dimensional micellar models of the colloidal stabilizations of the stabilized carbonate particles with different molecular structures of the surfactant. The diffusion of spin-probe molecules into the surfactant shells were studied by applying a starting force to push the molecules towards the carbonate surfaces and then observing the ensuing behavior. The simulations are in accordance with the ESR data and show that the geometrical organization of the surfactants depends on their molecular structure. The compactness of surfactant shells influences the properties of overbased detergents, especially their stability, their interaction with other additives used in lubricating oil formulations and with the acids produced by the combustion of fuel.

Direct electron paramagnetic resonance monitoring of the peptide synthesis coupling reaction in polymeric support

This work demonstrates, for the first time, a time-resolved electron paramagnetic resonance (EPR) monitoring of a chemical reaction occurring in a polymeric structure. The progress of the coupling of a Nα-tert-butyloxycarbonyl-2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (Boc-TOAC) spin probe to a model peptide-resin was followed through EPR spectra. Progressive line broadening of EPR peaks was observed, indicative of an increased population of immobilized spin probe molecules attached to the solid support. The time for spectral stabilization of this process coincided with that determined in a previous coupling study, thereby validating this in situ quantitative monitoring of the reaction. In addition, the influence of polymer swelling degree and solvent viscosity, as well as of the steric hindrance within beads, on the rate of coupling reaction was also addressed. A deeper evaluation of the latter effect was possible by determining unusual polymer parameters such as the average site–site distance and site-concentration within resin beads in each solvent system.

ESR Spin Probes in Ionic Liquids

The spin probes 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO), 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), and 2,2,6,6-tetramethyl-4-trimethylammoniumpiperidine-1-oxylIodide (CAT-1) are examined in a number of ionic liquids based on substituted imidazolium cations and tetrafluoroborate and hexafluorophosphate anions, respectively. The reorientation correlation times tau(R) of the spin probes in these systems have been determined by complete spectra simulation and, for rapid reortientation, by analysis of the intensities of the hyperfine lines of the electron spin resonance (ESR) spectra. A comparison of the results with those from the model system glycerol/water and selected organic solvents is made. Additions of diamagnetic and paramagnetic ions allow the conclusion that salt effects and spin exchange are present, and that both are superimposed by motional effects. Specific interactions in the ionic liquids, as well as between the spin-probe molecules and the constituents of the ionic liquids are reflected in the spectra of the spin probes, depending on their molecular structure.

The solution flow through the nanochannel of MCM-41: a spin-probe study

A spin-probe electron spin resonance (ESR) study was made on the alcoholic solution flowing through a quartz column packed with MCM-41 to clarify the dynamics of the liquid molecules in the nanochannel. The ESR spectra of a few hydrophobic spin probes showed that they undergo rotational diffusion preferentially along the longest molecular axis, indicating that the nanochannel is effectively narrowed further for these radicals by the influence of the solvent. Since almost identical ESR spectra were observed for the static samples, which were prepared in vacuo by introducing the solutions into the quartz tube with the MCM-41 powder and sealing off, the solution in the above-mentioned experiment should really flow through the nanochannel of MCM-41. Although a laminar flow is expected from the classical theory, the calculated flow rate is almost zero. In addition, the duration for the spin-probe molecules to flow through the column was basically not dependent on their affinity to the silica surface. To explain all these phenomena, we propose a model that the liquid molecules flow collectively by slipping on the surface of the nanochannel.

A direct simulation of EPR slow-motion spectra of spin labelled phospholipids in liquid crystalline bilayers based on a molecular dynamics simulation of the lipid dynamics

EPR line shapes can be calculated from the stochastic Liouville equation assuming a stochastic model for the reorientation of the spin probe. Here we use instead and for the first time a detailed molecular dynamics (MD) simulation to generate the stochastic input to the Langevin form of the Liouville equation. A 0.1 μs MD simulation at T = 50°C of a small lipid bilayer formed by 64 dipalmitoylphosphatidylcholine (DPPC) molecules at the water content of 23 water molecules per lipid was used. In addition, a 10 ns simulation of a 16 times larger system consisting of 32 DPPC molecules with a nitroxide spin moiety attached at the sixth position of the sn2 chain and 992 ordinary DPPC molecules, was used to investigate the extent of the perturbation caused by the spin probe. Order parameters, reorientational dynamics and the EPR FID curve were calculated for spin probe molecules and ordinary DPPC molecules. The timescale of the electron spin relaxation for a spin-moiety attached at the sixth carbon position of a DPPC lipid molecule is 11.9 × 107 rad s−1 and for an unperturbed DPPC molecule it is 3.5 × 107 rad s−1.

Libration motion of guest spin probe molecules in organic glasses: CW EPR and electron spin echo study

Echo-detected (ED) EPR spectra of nitroxide spin probes dissolved in glassy materials provide evidence that guest molecules in these media undergo fast librational motion. Theory of spin relaxation of a librating molecule is presented. The mean squared amplitude, 〈 α 2〉, of this motion which can be derived from continuous wave (CW) EPR spectral splitting is found to depend linearly on temperature in the low temperature region. This may be ascribed to thermal harmonic vibrations. The slope of the linear dependence varies from glass to glass and seems to correlate with the strength of the intermolecular bonds and with a degree of the fragility of the glass. Above the glass transition temperature 〈 α 2〉 increases sharply. Different applications are discussed: study of molecular properties of glass, intracellular glass formation in plant tissues, structural investigations.

Temperature dependence of amplitudes of libration motion of guest spin-probe molecules in organic glasses

Electron paramagnetic resonance (EPR) lineshapes of nitroxide spin probes in a variety of organic supercooled liquids and glasses were analyzed within the framework of the fast librational motion model. Computer simulation confirmed that the EPR lineshape may be fitted well by the librational model. For all systems studied, a mean-squared amplitude of this motion, 〈α2〉, was found to depend linearly on temperature in the low-temperature region, which is typical of harmonic solids. The slope of the linear dependence varies from glass to glass and does not change for different nitroxides in the same glass. This slope seems to correlate with the strength of the intermolecular bonds and with a degree of the fragility of the glass. Steep rises in 〈α2〉 were observed at high temperature.

Phase separation of mixed LB films containing spin probe molecules

Polyion complexed mixed Langmuir–Blodgett (LB) films of amphiphiles with long fluorocarbon chains (PFECA [CF 3(CF 2) 8(CH 2) 2O(CH 2) 2COOH] or perfluorodecanoic acid (PFDA) [CF 3(CF 2) 8COOH]) and those with long hydrocarbon chains (stearic acid (STA) or doxylstearic acids (5-, 10- or 16-doxyslstearic acid) deposited on Si, glass substrates or polyethylene terephthalate (PETP) sheets were characterized by atomic force microscopy/friction force microscopy (AFM/FFM) and electron spin resonance (ESR) spectroscopy. AFM/FFM images of 1:1 mixed LB films of PFECA (or PFDA) and 16-DXSTA showed clear phase-separated structure, while those of PFECA (or PFDA) and 5-DXSTA did not. All the values of the frictional force for the LB films, which did not show clear phase-separated structure, were intermediate between those for PFECA and STA. ESR spectra of mixed LB films of PFECA and 16-DXSTA were almost the same as that of a polyion complexed LB film of pure 16-DXSTA. On the contrary, the ESR spectra of mixed LB films of PFECA, STA and 5-DXSTA were very different from those of mixed LB films of 5-DXSTA and STA. From these results it was concluded that in the polyion complexed 1:1 mixed LB films of 5-DXSTA and PFECA, 5-DXSTA and PFDA, and 10-DXSTA and PFDA, the amphiphilic molecules with fluorocarbon chains and those with hydrocarbon chains are mixed with each other on the molecular level. This finding shows that phase separation of mixed LB films containing amphiphiles with fluorocarbon chains and those with hydrocarbon chains can be controlled by the selection of the structure of amphiphiles.

ESR study of the dynamics of adsorbed nitroxide radicals on porous surfaces in the dehydration process

The dynamics of 3-carbamoyl-2,2,5,5-tetramethyl-3-pyrrolin-1-yloxy (Tempyo), 4-oxo-2,2,6,6-tetramethyl-1-piperin-yloxy (4-oxo-Tempo) and 4-acetamide-2,2,6,6-tetramethyl-1-piperidin-yloxy (4-acetamide-Tempo) nitroxide radicals in aqueous solutions adsorbed on hydrophilic and hydrophobic SiO 2, Al 2O 3 and NaY zeolites with respect to dehydration degree were studied by ESR spectroscopy. The viscosity and the correlation times for the rotational motion of the adsorbed radicals depend on the dehydration degree, the nature of the support surfaces and the characteristics of the spin probe molecules.

Librational motion of guest spin probe molecules in glassy media

Echo-detected (ED) EPR spectra of nitroxide spin probes dissolved in glassy materials evidence that guest molecules in these media undergo fast librational motion. The mean square amplitude 〈 α 2〉 of this motion can be simply derived from continous wave (CW) EPR spectra by measuring the field separation between the two outer spectral peaks. By comparing the ED and CW EPR data the correlation time τ c of librational motion could be evaluated. Above the glass transition temperature 〈 α 2〉 was found to increase sharply.

Chain Mobility Restrictions in Random Ionomers Studied by Electron Spin Resonance Spectroscopy

Electron spin resonance (ESR) spectroscopy has been used to obtain information on chain mobility in spin-doped poly[styrene-co-(sodium methacrylate)] random ionomers. The results show that spin probe molecules randomly dispersed in ionomers respond to the matrix glass transition. However, when the probe molecules are anchored to the ionic multiplets, their mobility is reduced, indicating that the polymer segments adjacent to the ionic multiplets are constrained, and their mobility is restricted. Significant motions of the probe molecules start only when the kinetics of the ion-hopping process becomes relevant. Plasticization of the ionomers with polystyrene oligomers allowed for the simultaneous observation of two different mobility regimes, assigned to the matrix and cluster phases. These findings support the suggestion, which is a major component of the EHM model for the morphology of random ionomers, that regions of restricted mobility exist around the ionic multiplets.

Spin probe reduction in cells and tissues.

The reduction rates of different nitroxides in rat lung tissue were measured by electron paramagnetic resonance. We confirmed that the reduction rate of nitroxide spin probe molecules is coupled with their structure and transport characteristics. Oxygen was found to slow down the reduction rate of nitroxides in rat lung tissues. On the basis of these findings it can be concluded that most nitroxide properties described for homogeneous systems--cells and tissue homogenates--are also valid for heterogeneous systems, which is important for the application of nitroxides as metabolically active NMR contrast agents.

Chain mobility in solid polyelectrolyte complexes

Abstract2H NMR is used to study the mobility of the alkylene chain in solid poly[(dimethyliminio)‐alkylene]s (α,ω‐ionenes) complexed with poly(styrenesulfonate). In 10, 10‐ionene the alkylene chain motion was probed on samples selectively deuterated at the 1‐, 2‐, and 4‐position as well as in the methyl part of the quaternary ammonium groups. 2H NMR spectra show that all positions in the polymethylene chains are involved in conformational jumps between trans and gauche states. The mobility of the methylene units adjacent to charged centres was found to be significantly reduced compared with that of units in the inner part of the chain. The charged quaternary ammonium groups themselves, however, do not take part in trans‐gauche isomerization. An increase in mobility resulting from increasing the length of the alkylene chains between the charge centres was observed in 3,3‐, 6,6‐ and 10,10‐ionene complexes labelled in the 2‐position. The differences in chain dynamics were also detected via EPR line shapes of small spin probe molecules incorporated into the complexes.

Direct electron spin resonance evidence for α-tocopherol-induced phase separation in model membranes

The high resolution (narrow linewidth) amphiphilic spin probe perdeutero di- t-butyl nitroxide (PDDTBN) has been used to investigate the effect of α-tocopherol on lecithin liposomes. The electron spin resonance (ESR) results obtained as a function both of α-tocopherol concentration and of temperature indicate the presence of two different hydrophobic sites for the spin probe molecules. The presence of two distinct phases, one α-tocopherol-poor and the other α-tocopherol-rich, is suggested in these phospholipid bilayers.