Microscopic Order Macroscopic Disorder Research Articles

Theory and Least Squares Fitting of CW ESR Saturation Spectra Using the MOMD Model.

CW saturation experiments are widely used in ESR studies of relaxation processes in proteins and lipids. We develop the theory of saturation in ESR spectra in terms of its close relation with that of 2D-ELDOR. Our treatment of saturation is then based on the microscopic order macroscopic disorder (MOMD) model and can be used to fit the full CW saturation spectrum, rather than fitting just the peak-peak amplitude as a function of microwave field B 1 as is commonly done. This requires fewer experiments to yield effects on T 1, as well as provides a more extensive dynamic structural picture, for example, for scanning experiments on different protein sites. The code is released as a publicly available software package in Python that can be used to fit CW saturation spectra from biological samples of interest.

Influence of the Nature of the Ion Pairs on the Segmental Dynamics in Polyelectrolyte Complex Coacervate Phases

A spin label has been covalently attached to the weak polyanion poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the segmental dynamics of the polyanion backbone in polyelectrolyte complex coacervate phases formed with oppositely charged polycations such as poly(diallyldimethylammonium chloride) (PDADMAC), linear poly(ethylenimine) (l-PEI) and poly(allylamine hydrochloride) (PAH) by continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy. The segmental rotational mobility of the spin-labeled polyanion has been determined by the simulation of the line shape of the experimental CW EPR spectra using the microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion. The study has shown that the segmental mobility of the polyanion backbone in the coacervate phases decreases in the sequence PDADMAC > l-PEI > PAH. The nature of the ion pairs formed between the oppositely charged polyelectrolytes has a strong impact on the dynamics of the polyanion.

Protein Dynamics in the Solid State from (2)H NMR Line Shape Analysis. II. MOMD Applied to C-D and C-CD3 Probes.

Deuterium line shape analysis from mobile C–D and C–CD3 groups has emerged as a particularly useful tool for studying dynamics in the solid state. The theoretical models devised so far consist typically of sets of independent dynamic modes. Each such mode is simple and usually case-specific. In this scenario, model improvement entails adding yet another mode (thereby changing the overall model), comparison of different cases is difficult, and ambiguity is unavoidable. We recently developed the microscopic order macroscopic disorder (MOMD) approach as a single-mode alternative. In MOMD, the local spatial restrictions are expressed by an anisotropic potential, the local motion by a diffusion tensor, and the local molecular geometry by relative (magnetic and model-related) tensor orientations, all of adjustable symmetry. This approach provides a consistent method of analysis, thus resolving the issues above. In this study, we apply MOMD to PS-adsorbed LKα14 peptide and dimethylammonium tetraphenylborate (C–CD3 and N–CD3 dynamics, respectively), as well as HhaI methyltransferase target DNA and phase III of benzene-6-hexanoate (C–D dynamics). The success with fitting these four disparate cases, as well as the two cases in the previous report, demonstrates the generality of this MOMD-based approach. In this study, C–D and C–CD3 are both found to execute axial diffusion (rates R⊥ and R∥) in the presence of a rhombic potential given by the L = 2 spherical harmonics (coefficients c02 and c22). R⊥ (R∥) is in the 102–103 (104–105) s–1 range, and c02 and c22 are on the order of 2–3 kBT. Specific parameter values are determined for each mobile site. The diffusion and quadrupolar tensors are tilted at either 120° (consistent with trans–gauche isomerization) or nearly 110.5° (consistent with methyl exchange). Future prospects include extension of the MOMD formalism to include MAS, and application to 15N and 13C nuclei.

Rotational Dynamics of Spin-Labeled Polyacid Chain Segments in Polyelectrolyte Complexes Studied by CW EPR Spectroscopy

A nitroxide spin label has been covalently linked to the weak polyacid poly(ethylene-alt-maleic acid) (P(E-alt-MA)) to study the rotational mobility of the polyacid backbone in polyelectrolyte complexes (PEC) formed with the oppositely charged strong polycation poly(diallyldimethylammonium chloride) (PDADMAC) in dependence on the pH of the dispersion and the temperature. The rotational mobility of the polyacid chain segments has been determined by simulation of the line shape of the continuous wave (CW) electron paramagnetic resonance (EPR) spectra using the microscopic order/macroscopic disorder (MOMD) model of restricted rotational diffusion. The study has shown that the diffusion coefficient characterizing the rotational motions of the polyacid backbone is significantly smaller at low degree of dissociation at pH 4 than at high degree of dissociation at pH 7 and pH 10.

Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Structure and Dynamics of Lens Lipid Membranes Derived from a Single Porcine Donor: High Field EPR Study

To obtain correct EPR line shapes, spin-lattice relaxation times, and bimolecular collision rates between spin labels and oxygen, samples must be thoroughly deoxygenated or equilibrated with a controlled oxygen partial pressure. These measurements are conveniently carried out using a gas permeable plastic sample tube of small diameter that fits in a loop-gap resonator. Flow of gas over the tube allows easy deoxygenation or controlled oxygenation of the sample. In the initial design of the W-band (94 GHz) loop-gap resonator, samples were equilibrated with gas at room temperature outside the resonator, transferred to a quartz capillary, and positioned in the resonator. In a recently designed W-band loop-gap resonator, the sample is positioned in a gas-permeable Teflon tubing inside the resonator, which allows measurements of extremely small volume (∼30 nL) of sample. We used this new design to measure properties of lens lipid membranes derived from total lipids extracted from both lenses (single donor) of a 2-year-old porcine cortex and nucleus. Detailed profiles of membrane fluidity and oxygen transport parameter were obtained from saturation recovery EPR. Analysis of conventional spectra using the microscopic-order macroscopic-disorder (MOMD) model provided rotational diffusion coefficients (R(perpendicular) and R(parallel)) and order parameters. Three different types of motion of lipid spin labels n-PC, T-PC, and CSL (ASL) with, respectively, nitroxide z-axis, x-axis, and y-axis parallel to the bilayer normal, are discussed. Results demonstrate that EPR at W-band has the potential to be a powerful tool for studying samples of small volume, ∼30 nL, obtained from eye lenses of a single human donor.

Using spin-label W-band EPR to study membrane fluidity profiles in samples of small volume

Conventional and saturation-recovery (SR) EPR at W-band (94GHz) using phosphatidylcholine spin labels (labeled at the alkyl chain [n-PC] and headgroup [T-PC]) to obtain profiles of membrane fluidity has been demonstrated. Dimyristoylphosphatidylcholine (DMPC) membranes with and without 50mol% cholesterol have been studied, and the results have been compared with similar studies at X-band (9.4GHz) (L. Mainali, J.B. Feix, J.S. Hyde, W.K. Subczynski, J. Magn. Reson. 212 (2011) 418–425). Profiles of the spin-lattice relaxation rate (T1-1) obtained from SR EPR measurements for n-PCs and T-PC were used as a convenient quantitative measure of membrane fluidity. Additionally, spectral analysis using Freed’s MOMD (microscopic-order macroscopic-disorder) model (E. Meirovitch, J.H. Freed J. Phys. Chem. 88 (1984) 4995–5004) provided rotational diffusion coefficients (R⊥ and R||) and order parameters (S0). Spectral analysis at X-band provided one rotational diffusion coefficient, R⊥. T1-1, R⊥, and R|| profiles reflect local membrane dynamics of the lipid alkyl chain, while the order parameter shows only the amplitude of the wobbling motion of the lipid alkyl chain. Using these dynamic parameters, namely T1-1, R⊥, and R||, one can discriminate the different effects of cholesterol at different depths, showing that cholesterol has a rigidifying effect on alkyl chains to the depth occupied by the rigid steroid ring structure and a fluidizing effect at deeper locations. The nondynamic parameter, S0, shows that cholesterol has an ordering effect on alkyl chains at all depths. Conventional and SR EPR measurements with T-PC indicate that cholesterol has a fluidizing effect on phospholipid headgroups. EPR at W-band provides more detailed information about the depth-dependent dynamic organization of the membrane compared with information obtained at X-band. EPR at W-band has the potential to be a powerful tool for studying membrane fluidity in samples of small volume, ∼30nL, compared with a representative sample volume of ∼3μL at X-band.

Dynamics and ordering of lipid spin-labels along the coexistence curve of two membrane phases: An ESR study

An analysis of electron spin resonance (ESR) spectra from compositions along the liquid-ordered (Lo) and liquid-disordered (Ld) coexistence curve from the brain-sphingomyelin/dioleoylphosphatidylcholine/cholesterol (SPM/DOPC/Chol) model lipid system was performed to characterize the dynamic structure on a molecular level of these coexisting phases. We obtained 200 continuous-wave ESR spectra from glycerophospholipid spin-labels labeled at the 5, 7, 10, 12, 14, and 16 carbon positions of the 2nd acyl chain, a sphingomyelin spin-label labeled at the 14 carbon position of the amide-linked acyl chain, a headgroup-labeled glycerophospholipid, a headgroup-labeled sphingomyelin, and the cholesterol analogue spin-label cholestane all within multi-lamellar vesicle suspensions at room temperature. The spectra were analyzed using the MOMD (microscopic-order macroscopic-disorder) model to provide the rotational diffusion rates and order parameters which characterize the local molecular dynamics in these phases. The analysis also incorporated the known critical point and invariant points of the neighboring three-phase triangle along the coexistence curve. The variation in the molecular dynamic structures of coexisting Lo and Ld compositions as one moves toward the critical point is discussed. Based on these results, a molecular model of the Lo phase is proposed incorporating the “condensing effect” of cholesterol on the phospholipid acyl chain dynamics and ordering and the “umbrella model” of the phospholipid headgroup dynamics and ordering.

Electron Spin Resonance Investigation of Microscopic Viscosity, Ordering, and Polarity in Nafion Membranes Containing Methanol−Water Mixtures

Electron spin resonance (ESR) was used to monitor the local environment of 2,2,6,6-tetramethyl-4-piperidone N-oxide (Tempone) spin probe in water and methanol mixtures in solution and in Li(+) ion exchanged Nafion 117 membranes. Solution spectra were analyzed using the standard fast-motion line width parameters, while membrane spectra were fitted using the microscopic order macroscopic disorder (MOMD) slow-motional line shape program of Freed and co-workers. The (14)N hyperfine splitting, aN, which reflects the local polarity of the nitroxide probe, decreases with increasing methanol concentration, consistent with the decrease in solvent polarity. The polarity depended only weakly on composition in the Nafion membrane, but was noticeably more temperature-dependent. The microviscosity of the membrane aqueous phase as reflected by the rotational correlation time (tauc) of the probe, was nearly 2 orders of magnitude longer in the membrane than in solution and varied by an order of magnitude over the composition range studied. The probe exhibits significant local ordering in the aqueous phase of Nafion membranes that is diminished with increasing methanol concentration.

EPR spin label study of walnut oil effects on phosphatidylcholine membranes

Effects of walnut oil (WO) on dynamic and thermodynamic properties of 0–50 wt% cholesterol (CH) containing dimyristoylphosphatidylcholine (DMPC) and 10 wt% CH containing dipalmitoylphosphatidylcholine (DPPC) membrane dispersions were studied by electron paramagnetic resonance (EPR), using 5-doxyl stearic acid (5-DSA) and 16-doxyl stearic acid (16-DSA). Incorporation of 10 wt% WO alone decreased the phase transition temperature and created depth-dependent effects at the gel phase. The order increased close to the head region and decreased in the hydrocarbon core of the DMPC bilayer. For DPPC, the order decreased both close to head region and in the hydrocarbon core. Ten weight percent WO did not have considerable effect at the fluid phase for both DMPC and DPPC. Incorporation of 40 wt% WO into DMPC created an abrupt decrease in the maximum hyperfine splitting values after 305 K. The effect of 10 wt% WO in CH containing DMPC dispersions was dependent on the CH concentration. An increase and a decrease in the order were observed at low and high CH concentrations, respectively. Incorporation of WO created different effects on fluidity of 10 wt% CH containing DMPC and DPPC dispersions. Close to the head group region, the order in DMPC increased both in the gel and fluid phases; but for DPPC, an opposite effect was observed in both of the phases. In the hydrocarbon core of the bilayer, addition of 10 wt% WO into 10 wt% CH containing DMPC decreased the order in the gel phase and WO did not affect the order in the fluid phase. For DPPC, WO effects were observed to alter with temperature. In the studied temperature range, order parameters, diffusion constants and effective tilt angles were obtained from simulations of the spectra using Microscopic Order Macroscopic Disorder (MOMD) and Vary Anisotropic Reorientation (VAR) models. For 16-DSA, spectra were also simulated using two domains with EPRSIM.

An Assessment of the Applicability of Multifrequency ESR to Study the Complex Dynamics of Biomolecules

It is shown that the commonly used models for analyzing ESR spectra from nitroxide spin-labeled proteins or DNA systems are special cases of the more general slowly relaxing local structure (SRLS) model, wherein the nitroxide spin probe is taken as reorienting in a restricted local environment, which itself is relaxing on a longer time scale. This faster motion describes the internal dynamics, while the slower motion describes the global tumbling of the macromolecule. By using the SRLS model as the reference, it is shown (1) under what conditions the microscopic-order macroscopic-disorder (MOMD) model, wherein the global tumbling of the macromolecule is in the rigid limit, is valid, and (2) when the fast internal motion (FIM) model, wherein the internal motion is so rapid as to lead to partial averaging of the magnetic tensors, is valid. The frequency dependence of these models is studied. A key general property of high frequency ESR that emerges is that it reports on a faster motional time scale, whereas low frequency ESR reports on a slower motional time scale. It is shown that, in general, the MOMD model is a better approximation for ESR spectra obtained at high frequency (250 GHz), whereas, in general, the FIM model is a better approximation for low frequency (9 GHz) ESR spectra. However, in general, one does not find that the simpler model fits, at a single ESR frequency, to the more complete SRLS model, return correct motional and ordering parameters. The simultaneous fitting of both low and high frequency ESR spectra is thus required to remove such ambiguities and to return all the various dynamic, ordering, and geometric factors that characterize the complex dynamics. This approach is briefly related to recent ESR spectra from the spin-labeled protein, T4 lysozyme, and from spin-labeled DNA nucleosides. In order to better apply the slow-motional SRLS model to macromolecular dynamics, the Polimeno-Freed theory has been extended to the case where the global tumbling is anisotropic and where the angle between the principal axis of the global motion and the preferred orientation of the internal modes of motion is arbitrary.

A quantitative electron spin resonance line shape study of the order-disorder transition in the lamellar phase of the palmitoyllysophosphatidylcholine-water system

A quantitative slow-motional ESR line shape study is reported of the lamellar phase and the micellar phase of the palmitoyllysophosphatidylcholine (PaLPC)-water system. The lamellar spectra are simulated using the microscopic order-macroscopic disorder (MOMD) model developed by Meirovitch, E., Nayeem, A., and Freed, J. H., 1984, J. phys. Chem., 88, 3454, whereas the micellar spectra are interpreted in terms of a two-dynamics model developed in this laboratory (Liang, Z., and Westlund, P.-O., 1993, J. chem. Phys., 99, 7098). For the lamellar phase, an order-disorder transition is observed from the temperature dependence of the spectra and quantized in terms of the model parameters, yielding a transition temperature of about 45°C. A comparison of the model parameters is made between the lamellar and the micellar phases, which indicates a phase independence of the local orientational order. A similar comparison is made also between the oleoyllysophospatidylcholine-water and the PaLPC-water systems in the lam...