Resonance Line Shape Analysis Research Articles

NMR lineshape analysis using analytical solutions of multi-state chemical exchange with applications to kinetics of host–guest systems

Nuclear magnetic resonance (NMR) lineshape analysis is a powerful tool for the study of chemical kinetics. Here we provide techniques for analysis of the relationship between experimentally observed spin kinetics (transitions between different environments A,B,dots) and corresponding chemical kinetics (transitions between distinct chemical species; e.g., free host and complexed host molecule). The advantages of using analytical solutions for two-, three- or generally N-state exchange lineshapes (without J-coupling) over the widely used numerical calculation for NMR spectral fitting are presented. Several aspects of exchange kinetics including the generalization of coalescence conditions in two-state exchange, the possibility of multiple processes between two states, and differences between equilibrium and steady-state modes are discussed. ‘Reduced equivalent schemes’ are introduced for spin kinetics containing fast-exchanging states, effectively reducing the number of exchanging states. The theoretical results have been used to analyze a host–guest system containing an oxoporphyrinogen complexed with camphorsulfonic acid and several other literature examples, including isomerization, protein kinetics, or enzymatic reactions. The theoretical treatment and experimental examples present an expansion of the systematic approach to rigorous analyses of systems with rich chemical kinetics through NMR lineshape analysis.

Studying Structural and Dynamic Properties of KCNE3 in Various Membrane Environments Using Molecular Dynamics Simulation and EPR Spectroscopy

KCNE3 is a single transmembrane protein that modulates the function and trafficking of several voltage-gated potassium channels, including KCNQ1, KCNQ4, hERG, Kv2.1, Kv3.1, and Kv3.2. KCNE3 binds with KCNQ1 and plays a crucial role in potassium ion recycling, which is important for transepithelial chloride ion secretion. Mutations in KCNE3 are linked to diseases associated with salt and fluid disorders including cystic fibrosis (CF). Despite the biological significance, structural and dynamic properties of KCNE3 is not fully understood in different membrane environment. In this study, we investigate structural and dynamic properties of KCNE3 in different membrane environments such as POPC, POPC/POPG, DMPC, DOPC, using molecular dynamics simulation. The amino acid locations and side chain dynamics of WT KCNE3 will be compared to the mutated form of KCNE3. The molecular dynamics simulation studies will be compared with the continuous wave electron paramagnetic resonance (CW-EPR) lineshape analysis results.

Complexation Kinetics of Cyclodextrins with Bile Salt Anions: Energy Barriers for the Threading of Ionic Groups.

Binding constants for thousands of cyclodextrin complexes have been reported in the literature, but much less is known about the kinetics of these host-guest complexes. In the present study, inclusion complexes of bile salts with β-cyclodextrin, γ-cyclodextrin, and a methylated β-cyclodextrin were studied by nuclear magnetic resonance (NMR) lineshape analysis to explore the structural factors that govern the complexation kinetics. For complexes with β-cyclodextrin, the association rate constants ranged from 2 × 106 to 2 × 107 M-1 s-1 while the dissociation rate constants ranged from 12 to 6000 s-1 at 25 °C. The kinetics were thus significantly slower than for any other β-cyclodextrin complex reported in the literature, due to the large energy barrier for threading the ionic sidechains of the bile salt anions. Bile salts with taurine and glycine sidechains had identical binding affinities, but the kinetics differed by a factor of 10. Introduction of a single hydroxyl group at the binding site of the bile salts reduced the lifetimes and binding constants of the complexes by more than 50 times. The strong temperature dependence of the rate constants revealed that the large activation energies were mainly enthalpic with a small contribution from entropy. The larger γ-cyclodextrin was threaded by the nonionic end of the bile salts, and the kinetics were too fast to be accurately determined. The study demonstrates that ionic groups on guest molecules constitute significant energy barriers for the threading and dethreading of β-cyclodextrin hosts.

Biradical rotamer states tune electron J coupling and MAS dynamic nuclear polarization enhancement

Cross Effect (CE) Dynamic Nuclear Polarization (DNP) relies on the dipolar (D) and exchange (J) coupling interaction between two electron spins. Until recently only the electron spin D coupling was explicitly included in quantifying the DNP mechanism. Recent literature discusses the potential role of J coupling in DNP, but does not provide an account of the distribution and source of electron spin J coupling of commonly used biradicals in DNP. In this study, we quantified the distribution of electron spin J coupling in AMUPol and TOTAPol biradicals using a combination of continuous wave (CW) X-band electron paramagnetic resonance (EPR) lineshape analysis in a series of solvents and at variable temperatures in solution – a state to be vitrified for DNP. We found that both radicals show a temperature dependent distribution of J couplings, and the source of this distribution to be conformational dynamics. To qualify this conformational dependence of J coupling in both molecules we carry out Broken Symmetry DFT calculations which show that the biradical rotamer distribution can account for a large distribution of J couplings, with the magnitude of J coupling directly depending on the relative orientation of the electron spin pair. We demonstrate that the electron spin J couplings in both AMUPol and TOTAPol span a much wider distribution than suggested in the literature. We affirm the importance of electron spin J coupling for DNP with density matrix simulations of DNP in Liouville space and under magic angle spinning, showcasing that a rotamer with high J coupling and optimum relative g-tensor orientation can significantly boost the DNP performance compared to random orientations of the electron spin pair. We conclude that moderate electron spin J coupling above a threshold value can facilitate DNP enhancements.

Characterizing the structure of styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) using RAFT polymerization for membrane protein spectroscopic studies.

Membrane proteins play an important role in maintaining the structure and physiology of an organism. Despite their significance, spectroscopic studies involving membrane proteins remain challenging due to the difficulties in mimicking their native lipid bilayer environment. Membrane mimetic systems such as detergent micelles, liposomes, bicelles, nanodiscs, lipodisqs have improved the solubility and folding properties of the membrane proteins for structural studies, however, each mimetic system suffers from its own limitations. In this study, using three different lipid environments, vesicles were titrated with styrene-maleic acid (StMA) copolymer leading to a homogeneous SMALP system (∼10 nm) at a weight ratio of 1:1.5 (vesicle: StMA solution). A combination of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) was used to characterize these SMALPs. We used a controlled synthesis mechanism to synthesize StMA based block copolymers called reversible addition-fragmentation chain transfer polymerization (RAFT) SMALPs. Incorporation of the Voltage Sensor Domain of KCNQ1 (Q1-VSD) into RAFT SMALPs indicates that this is a promising application of this system to study membrane proteins using different biophysical techniques. V165C in Q1-VSD corresponding to the hydrophobic region was incorporated into the SMALP system. Continuous Wave-Electron Paramagnetic Resonance (CW-EPR) line shape analysis showed line shape broadening, exposing a lower rigid component and a faster component of the spin label.

Electron spin resonance studies on deuterated nitroxyl spin probes used in Overhauser-enhanced magnetic resonance imaging.

The electron spin resonance studies were carried out for 2mm concentration of 14 N-labeled and 15 N-labeled 3-carbamoyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl, 3-carboxy-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl, 3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine-1-oxyl and their deuterated nitroxyl radicals using X-band electron spin resonance spectrometer. The electron spin resonance line shape analysis was carried out. The electron spin resonance parameters such as linewidth, Lorentzian component, signal intensity ratio, rotational correlation time, hyperfine coupling constant and g-factor were estimated. The deuterated nitroxyl radicals have narrow linewidth and an increase in Lorentzian component, compared with undeuterated nitroxyl radicals. The dynamic nuclear polarization factor was observed for all nitroxyl radicals. Upon 2 H labeling, about 70% and 40% increase in dynamic nuclear polarization factor were observed for 14 N-labeled and 15 N-labeled nitroxyl radicals, respectively. The signal intensity ratio and g-value indicate the isotropic nature of the nitroxyl radicals in pure water. Therefore, the deuterated nitroxyl radicals are suitable spin probes for in vivo/in vitro electron spin resonance and Overhauser-enhanced magnetic resonance imaging modalities. Copyright © 2017 John Wiley & Sons, Ltd.

Two-Dimensional NMR Lineshape Analysis.

NMR titration experiments are a rich source of structural, mechanistic, thermodynamic and kinetic information on biomolecular interactions, which can be extracted through the quantitative analysis of resonance lineshapes. However, applications of such analyses are frequently limited by peak overlap inherent to complex biomolecular systems. Moreover, systematic errors may arise due to the analysis of two-dimensional data using theoretical frameworks developed for one-dimensional experiments. Here we introduce a more accurate and convenient method for the analysis of such data, based on the direct quantum mechanical simulation and fitting of entire two-dimensional experiments, which we implement in a new software tool, TITAN (TITration ANalysis). We expect the approach, which we demonstrate for a variety of protein-protein and protein-ligand interactions, to be particularly useful in providing information on multi-step or multi-component interactions.

Cholesterol enhances surface water diffusion of phospholipid bilayers.

Elucidating the physical effect of cholesterol (Chol) on biological membranes is necessary towards rationalizing their structural and functional role in cell membranes. One of the debated questions is the role of hydration water in Chol-embedding lipid membranes, for which only little direct experimental data are available. Here, we study the hydration dynamics in a series of Chol-rich and depleted bilayer systems using an approach termed (1)H Overhauser dynamic nuclear polarization (ODNP) NMR relaxometry that enables the sensitive and selective determination of water diffusion within 5-10 Å of a nitroxide-based spin label, positioned off the surface of the polar headgroups or within the nonpolar core of lipid membranes. The Chol-rich membrane systems were prepared from mixtures of Chol, dipalmitoyl phosphatidylcholine and/or dioctadecyl phosphatidylcholine lipid that are known to form liquid-ordered, raft-like, domains. Our data reveal that the translational diffusion of local water on the surface and within the hydrocarbon volume of the bilayer is significantly altered, but in opposite directions: accelerated on the membrane surface and dramatically slowed in the bilayer interior with increasing Chol content. Electron paramagnetic resonance (EPR) lineshape analysis shows looser packing of lipid headgroups and concurrently tighter packing in the bilayer core with increasing Chol content, with the effects peaking at lipid compositions reported to form lipid rafts. The complementary capability of ODNP and EPR to site-specifically probe the hydration dynamics and lipid ordering in lipid membrane systems extends the current understanding of how Chol may regulate biological processes. One possible role of Chol is the facilitation of interactions between biological constituents and the lipid membrane through the weakening or disruption of strong hydrogen-bond networks of the surface hydration layers that otherwise exert stronger repulsive forces, as reflected in faster surface water diffusivity. Another is the concurrent tightening of lipid packing that reduces passive, possibly unwanted, diffusion of ions and water across the bilayer.

Isomerase-catalyzed binding of interleukin-1 receptor-associated kinase 1 to the EVH1 domain of vasodilator-stimulated phosphoprotein.

Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168–Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.

Trace gas absorption spectroscopy using functionalized microring resonators

We detect trace gases at parts-per-billion levels using evanescent-field absorption spectroscopy in silicon nitride microring resonators coated with a functionalized sorbent polymer. An analysis of the microring resonance line shapes enables a measurement of the differential absorption spectra for a number of vapor-phase analytes. The spectra are obtained at the near-infrared overtone of OH-stretch resonance, which provides information about the toxicity of the analyte vapor.

Asymmetric Collapse in Biomimetic Complex Coacervates Revealed by Local Polymer and Water Dynamics

Complex coacervation is a phenomenon characterized by the association of oppositely charged polyelectrolytes into micrometer-scale liquid condensates. This process is the purported first step in the formation of underwater adhesives by sessile marine organisms, as well as the process harnessed for the formation of new synthetic and protein-based contemporary materials. Efforts to understand the physical nature of complex coacervates are important for developing robust adhesives, injectable materials, or novel drug delivery vehicles for biomedical applications; however, their internal fluidity necessitates the use of in situ characterization strategies of their local dynamic properties, capabilities not offered by conventional techniques such as X-ray scattering, microscopy, or bulk rheological measurements. Herein, we employ the novel magnetic resonance technique Overhauser dynamic nuclear polarization enhanced nuclear magnetic resonance (DNP), together with electron paramagnetic resonance (EPR) line shape analysis, to concurrently quantify local molecular and hydration dynamics, with species- and site-specificity. We observe striking differences in the structure and dynamics of the protein-based biomimetic complex coacervates from their synthetic analogues, which is an asymmetric collapse of the polyelectrolyte constituents. From this study we suggest charge heterogeneity within a given polyelectrolyte chain to be an important parameter by which the internal structure of complex coacervates may be tuned. Acquiring molecular-level insight to the internal structure and dynamics of dynamic polymer complexes in water through the in situ characterization of site- and species-specific local polymer and hydration dynamics should be a promising general approach that has not been widely employed for materials characterization.

Classification of breast lesions pre‐contrast injection using water resonance lineshape analysis

Inhomogeneously broadened, non-Lorentzian water resonances have been observed in small image voxels of breast tissue. The non-Lorentzian components of the water resonance are probably produced by bulk magnetic susceptibility shifts caused by dense, deoxygenated tumor blood vessels (the 'blood oxygenation level-dependent' effect), but can also be produced by other characteristics of local anatomy and physiology, including calcifications and interfaces between different types of tissue. Here, we tested the hypothesis that the detection of non-Lorentzian components of the water resonance with high spectral and spatial resolution (HiSS) MRI allows the classification of breast lesions without the need to inject contrast agent. Eighteen malignant lesions and nine benign lesions were imaged with HiSS MRI at 1.5 T. A new algorithm was developed to detect non-Lorentzian (or off-peak) components of the water resonance. After a Lorentzian fit had been subtracted from the data, the largest peak in the residual spectrum in each voxel was identified as the major off-peak component of the water resonance. The difference in frequency between these off-peak components and the main water peaks, and their amplitudes, were measured in malignant lesions, benign lesions and breast fibroglandular tissue. Off-peak component frequencies were significantly different between malignant and benign lesions (p < 0.001). Receiver operating characteristic (ROC) analysis was used to assess the diagnostic performance of HiSS off-peak component analysis compared with dynamic contrast-enhanced (DCE) MRI parameters. The areas under the ROC curves for the 'DCE rapid uptake fraction', 'DCE washout fraction', 'off-peak component amplitude' and 'off-peak component frequency' were 0.75, 0.83, 0.50 and 0.86, respectively. These results suggest that water resonance lineshape analysis performs well in the classification of breast lesions without contrast injection and could improve the diagnostic accuracy of clinical breast MR examinations. In addition, this approach may provide an alternative to DCE MRI in women who are at risk for adverse reactions to contrast media.

Sulfamate proton solvent exchange in heparin oligosaccharides: Evidence for a persistent hydrogen bond in the antithrombin-binding pentasaccharide Arixtra

Sulfamate groups (NHSO(3)(-)) are important structural elements in the glycosaminoglycans (GAGs) heparin and heparan sulfate (HS). In this work, proton nuclear magnetic resonance (NMR) line-shape analysis is used to explore the solvent exchange properties of the sulfamate NH groups within heparin-related mono-, di-, tetra- and pentasaccharides as a function of pH and temperature. The results of these experiments identified a persistent hydrogen bond within the Arixtra (fondaparinux sodium) pentasaccharide between the internal glucosamine sulfamate NH and the adjacent 3-O-sulfo group. This discovery provides new insights into the solution structure of the Arixtra pentasaccharide and suggests that 3-O-sulfation of the heparin N-sulfoglucosamine (GlcNS) residues pre-organize the secondary structure in a way that facilitates binding to antithrombin-III. NMR studies of the GlcNS NH groups can provide important information about heparin structure complementary to that available from NMR spectral analysis of the carbon-bound protons.

Nuclear quadrupole resonance lineshape analysis for different motional models: Stochastic Liouville approach

A general theory of lineshapes in nuclear quadrupole resonance (NQR), based on the stochastic Liouville equation, is presented. The description is valid for arbitrary motional conditions (particularly beyond the valid range of perturbation approaches) and interaction strengths. It can be applied to the computation of NQR spectra for any spin quantum number and for any applied magnetic field. The treatment presented here is an adaptation of the "Swedish slow motion theory," [T. Nilsson and J. Kowalewski, J. Magn. Reson. 146, 345 (2000)] originally formulated for paramagnetic systems, to NQR spectral analysis. The description is formulated for simple (Brownian) diffusion, free diffusion, and jump diffusion models. The two latter models account for molecular cooperativity effects in dense systems (such as liquids of high viscosity or molecular glasses). The sensitivity of NQR slow motion spectra to the mechanism of the motional processes modulating the nuclear quadrupole interaction is discussed.

A quick diagnostic test for NMR receiver gain compression

Modern NMR spectrometers require receivers to work within their linear ranges to maintain high fidelity line shape and peak integration. For better sensitivity, the receiver gain has to be optimized to detect dilute analytes; however, gain compression needs to be avoided. Here, we explore if and how linear receiver performance can be achieved for a couple of representative gain settings on a spectrometer. In the case of slight receiver gain compression, not only will the peak integral be attenuated but a very small line-shape change can also be observed. Hence, we can resort to resonance integration and line-shape analysis for gain compression diagnosis. As such, NMR signals, regardless of their observed amplitude difference in frequency domain, can be accurately compared in quantitative analysis.

ChemInform Abstract: Magnetic Resonance Studies of Ammonia Adsorption and Decomposition on Titania-Supported Vanadia Catalysts

In-situ proton and deuterion single-resonance NMR techniques were used to study the adsorption and decomposition of ammonia as a function of temperature on a series of V{sub 2}O{sub 5}/TiO{sub 2} catalysts with the weight loading of vanadia ranging from 0 to 6.1 wt % (approximately one monolayer). After analysis of magnetic resonance line shapes, several different molecular species were found to be present on the catalyst surfaces. At low adsorption temperatures on the low-loading samples, the predominant adsorbed species was intact, coordinated ammonia rotating about its C{sub 3} axis. As the weight loading or adsorption temperature was increased, the adsorbed, intact ammonia exhibited an increased H-N-H angle; the total number of protons associated with intact ammonia per gram of sample decreased. At the point where intact ammonia was no longer observed, a new species was observed and assigned to NH{sub 2} moieties. On all samples, ammonium ions were observed at low adsorption temperatures (100{degrees}C) but were found to be easily removed by heating. Their concentration was greater on the higher weight loading samples. The formation of short-lived VOH groups from the protons of dissociated NH{sub 3} was inferred on the higher weight loading samples. Multiple-quantum spin-counting experiments were done tomore » further investigate the nature of the adsorbed species and their groupings on the surfaces. 45 refs., 12 figs., 2 tabs.« less

Contactless conductivity of nanoparticles from electron magnetic resonance lineshape analysis

A contactless method to determine the electrical conductivity of nanoparticles is presented. It is based on the lineshape analysis of electron magnetic resonance signals which are ‘Dysonian’ for conducting samples of sizes larger than the skin depth. The method is validated by measurements on a bulk sample of La 0.67Sr 0.33MnO 3 where it gives values close to those obtained from direct measurement of conductivity and is then used to determine the conductivity of nanoparticles of La 0.67Sr 0.33MnO 3 dispersed in polyvinyl alcohol as a function of temperature.

Resonance structure in electron–N2 scattering around 11.5 eV: high-resolution measurements, ab initio calculations and line shape analyses

Using two different experimental setups with energy widths of about 6 and 13 meV, we obtained significantly improved results for the energy dependence of angle-differential (10°–180°) elastic and vibrationally inelastic cross sections for electron scattering from N2 molecules in the energy range around the narrow N−2(R2Σ+g) resonance. The energy location and the natural width of this resonance are determined as 11.497(2) eV and 1.3(2) meV, respectively. Ab initio potential energy curves are obtained from CCSD(T) calculations for the neutral N2(X1Σ+g) and N*2(E3Σ+g) states as well as for the N−2(R2Σ+g) resonance state. They corroborate quite accurately the measured resonance energy and provide accurate energy spacings and overlap integrals for the pertinent vibrational states. A detailed analysis of resonance line shapes for selected scattering angles is performed by applying a model for the interference of resonant and nonresonant scattering processes. It provides a link between the resonance width to absolute DCS and describes elastic and vibrational excitation processes on a common basis. Through both their size and sign, vibrational overlap integrals are shown to determine the observed Fano-type line shapes and account for the opposite asymmetries and intensity changes of adjacent vibrational resonance peaks. Fine-tuning of the fits to the observed shapes is achieved by proper parametrization of the nonresonant amplitudes. A highly resolved excitation function for the formation of the metastable N*2(E3Σ+g) level from threshold (near 11.88 eV) to 13.4 eV is also presented.

Fast-passage microwave molecular spectroscopy with frequency sweeping

We propose a new method of molecular microwave spectroscopy together with analysis of resonance line shapes, based on the fast passage effect. The fast passage effect is the excitation of coherent spontaneous radiation of tested molecules using a fast frequency sweep over the absorption line. We show that the line shape can be reconstructed from the received detector signal with the use of Hilbert and Fourier transformations. We find that sensitivity of a fast passage spectrometer is higher than that of an ordinary pulsed Fourier-transform spectrometer. The proposed method is a useful tool for fast analysis of gas mixtures.

Critical chain dynamics near the interdigitated–noninterdigitated chain configurational phase transition in decylammonium chloride

We have carried out a H1 nuclear magnetic resonance line shape analysis near the irreversible interdigitated-to-noninterdigitated chain configurational phase transition in decylammonium chloride (C10H21NH3Cl), a model biomembrane. The splitting parameter corresponding to the nematic order of the hydrocarbon chain shows a two-dimensional Ising behavior and indicates a chain defect motion in addition to the chain reorientational motion. The origin of the low-frequency critical fluctuation is attributed to a critical slowing down of the collective gear-like reorientation of the rigid hydrocarbon chain.