NMR Relaxation Studies Research Articles

The Dynamics of Dendrimers by NMR Relaxation: Interpretation Pitfalls

NMR is a powerful tool to study the dynamics of dendrimers. By analogy to linear polymers, shorter T(1) relaxation times have been traditionally associated to less mobile nuclei and hence, dendrimers described with reduced local motions at either the core or the periphery. Herein we report a NMR relaxation study [(1)H and (13)C T(1), T(2); (13)C{(1)H}NOE; various fields and temperatures] which reveals profound differences between the relaxation behavior of dendrimers and linear polymers. Dendrimers show slower dynamics at internal layers and on increasing generation and may display internal nuclei in the slow motional regime with larger T(1) values than the periphery. In contrast to the relaxation properties of linear polymers, these T(1) increments should not be interpreted as resulting from faster dynamics. Only the recording of T(1) data at various temperatures (alternatively, T(2) or NOE at one temperature) ensures the correct interpretation of dendrimer dynamics.

Frequency‐dependent NMR relaxation of liquids confined inside porous media containing an increased amount of magnetic impurities

Frequency-dependent NMR relaxation studies have been carried out on water (polar) and cyclohexane (nonpolar) molecules confined inside porous ceramics containing variable amounts of iron oxide (III). The porous ceramics were prepared by compression of powders mixed with iron oxide followed by thermal treatment. The pore size distribution was estimated using a technique based on diffusion in internal fields that exposed a narrow distribution of macropore sizes with an average pore dimension independent of iron oxide content. The relaxation dispersion curves were obtained at room temperature using a fast field cycling NMR instrument. They display an increase of the relaxation rate proportional to the iron oxide concentration. This behavior is more prominent at low Larmor frequencies and is independent of the polar character of the confined molecules. The results reported here can be fitted well with a relaxation model considering exchange between molecules in the close vicinity of the paramagnetic centers located in the surface and bulk-like molecules inside the pores. This model allows the extraction of the transverse diffusional correlation time that can be related to the polar character of the confined molecules.

Suppression of Cooperative Motions in Phospholipid Membranes by Osmotic Stress: Deuterium NMR Relaxation Study

Understanding membrane dynamics is crucial to explaining the function of membrane proteins. Phospholipids are commonly employed as model systems to investigate biological membranes. The complex dynamic organization of phospholipid membranes spans several frequency decades, starting from sub-picosecond local motions to millisecond collective dynamics [1]. Such motional frequencies can be accessed using various NMR relaxation methods. To address membrane dynamics mediated by osmotic stress, we measured 2H longitudinal (R1Z) and transverse quadrupolar echo (R2QE) relaxation rates for the liquid-crystalline phase of DMPC-d54 membrane bilayers. Osmotic stress was applied by both dehydration and osmolyte concentration [2]. The R1Z values of individual acyl segments were independent of osmotic stress while the segmental order parameters (SCD) and R1Z profiles followed a theoretical square-law functional dependence [3]. The R2QE rates were found to be sensitive to osmotic pressure as well as the acyl position, thus yielding two important observations: enhanced transverse relaxation rates with increased amount of water per lipid, and limiting lower R2QE values as we dehydrate the membrane. The R2QE rates of the acyl segments and respective SCD values tend to follow a square-law behavior [3] with increasing lipid dehydration. At higher hydration the square-law behavior is limited to those acyl segments deeper in the hydrophobic region, with a break as the head group is approached. These results clearly indicate that water enhances slow cooperative motions whereas they are suppressed by dehydration. Additional complementary Carr-Purcell-Meiboom-Gill (CPMG) dispersion measurements map the frequency dependence of relaxation rates. Such studies in presence of membrane proteins give insight into optimized lipid hydration for their biological functions.[1] A. Leftin et al. (2011) BBA 1808, 818-839.[2] K.J. Mallikarjunaiah et al. (2011) BJ100, 98-107.[3] M.F. Brown (1982) JCP77, 1576-1599.

Dynamics of the Influenza Hemagglutinin Fusion Peptide: A Comparison of MD and NMR

Membrane insertion of influenza hemagglutinin (HA) is essential for viral membrane fusion. Previous NMR relaxation studies on bicelles [1, 2] analyzed the 23 conserved N-terminal residues of the HA2 subunit (fusion peptide). It was shown that this peptide, which adopted a helical hairpin structure, experiences wobbling motions relative to the bilayer surface on the ns timescale. Here, the dynamics of the HA fusion peptide hairpin on a DMPC membrane are studied using molecular dynamics (MD) simulations. Simulations were performed with the acidic groups (E11 and D19) protonated and unprotonated. Internal correlation functions of backbone N-H vectors are determined over the 100-ns MD simulations, and are fit to the Lipari-Szabo model free approach [3]. The calculated order parameters and correlation times are similar to those determined experimentally. Starting from an initial orientation parallel to the membrane, during the simulations the hairpin rotated nearly 90° around the axis that is parallel to the two helices, with the N-terminal helix buried more deeply in the lipid tail region.[1] J. L. Lorieau, J. M. Louis, and A. Bax, Proceedings of the National Academy of Sciences of the United States of America 107 (2010) 11341.[2] J. L. Lorieau, J. M. Louis, and A. Bax, Journal of the American Chemical Society 133 (2011) 14184.[3] G. Lipari and A. Szabo, Biophysical Journal 37 (1982) A380.

13C NMR relaxation and computational study of anisole and derivatives in the solution state

13C NMR spin–lattice relaxation times and nuclear Overhauser effects were measured at several temperatures for the methoxyl methyl carbon and the phenyl ring carbons in neat samples and in dilute cyclohexane solution for anisole, 4‐methylanisole, and 4‐chloroanisole. Similar measurements were made for 2‐methylanisole, 2‐methyl‐4‐bromoanisole, and 2,4,6‐trimethylanisole in dilute cyclohexane solution. Density functional theory (DFT) computations were performed on anisole, 4‐chloroanisole and 2,4,6‐trimethylanisole to obtain the minimum energy structures and the potential energy barriers to the internal rotations of the methoxyl group. The shortest distance between a methoxyl methyl hydrogen and the ortho hydrogen in anisole is 1.920 Å. The DFT results point to steric interactions that arise thereof as the principal source of the energy barriers to the internal rotation of the methyl or of the methoxyl group. The carbon relaxation data are consistent with the existence of noncovalent intermolecular interaction, especially π − π stacking interaction. The nuclear magnetic resonance and DFT results are discussed with reference to the rotational characteristics of the methoxyl methyl and the anisotropy in the reorientational motion of anisole and its derivatives in dilute cyclohexane solution. Copyright © 2012 John Wiley & Sons, Ltd.

Accurate Prediction of the Dynamical Changes within the Second PDZ Domain of PTP1e

Experimental NMR relaxation studies have shown that peptide binding induces dynamical changes at the side-chain level throughout the second PDZ domain of PTP1e, identifying as such the collection of residues involved in long-range communication. Even though different computational approaches have identified subsets of residues that were qualitatively comparable, no quantitative analysis of the accuracy of these predictions was thus far determined. Here, we show that our information theoretical method produces quantitatively better results with respect to the experimental data than some of these earlier methods. Moreover, it provides a global network perspective on the effect experienced by the different residues involved in the process. We also show that these predictions are consistent within both the human and mouse variants of this domain. Together, these results improve the understanding of intra-protein communication and allostery in PDZ domains, underlining at the same time the necessity of producing similar data sets for further validation of thses kinds of methods.

NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions

Lytic polysaccharide monooxygenases currently classified as carbohydrate binding module family 33 (CBM33) and glycoside hydrolase family 61 (GH61) are likely to play important roles in future biorefining. However, the molecular basis of their unprecedented catalytic activity remains largely unknown. We have used NMR techniques and isothermal titration calorimetry to address structural and functional aspects of CBP21, a chitin-active CBM33. NMR structural and relaxation studies showed that CBP21 is a compact and rigid molecule, and the only exception is the catalytic metal binding site. NMR data further showed that His28 and His114 in the catalytic center bind a variety of divalent metal ions with a clear preference for Cu(2+) (K(d) = 55 nM; from isothermal titration calorimetry) and higher preference for Cu(1+) (K(d) ∼ 1 nM; from the experimentally determined redox potential for CBP21-Cu(2+) of 275 mV using a thermodynamic cycle). Strong binding of Cu(1+) was also reflected in a reduction in the pK(a) values of the histidines by 3.6 and 2.2 pH units, respectively. Cyanide, a mimic of molecular oxygen, was found to bind to the metal ion only. These data support a model where copper is reduced on the enzyme by an externally provided electron and followed by oxygen binding and activation by internal electron transfer. Interactions of CBP21 with a crystalline substrate were mapped in a (2)H/(1)H exchange experiment, which showed that substrate binding involves an extended planar binding surface, including the metal binding site. Such a planar catalytic surface seems well-suited to interact with crystalline substrates.

Understanding the Solvent Effect on the Catalytic Oxidation of 1,4‐Butanediol in Methanol over Au/TiO2 Catalyst: NMR Diffusion and Relaxation Studies

The effect of water on the catalytic oxidation of 1,4-butanediol in methanol over Au/TiO(2) has been investigated by catalytic reaction studies and NMR diffusion and relaxation studies. The addition of water to the dry catalytic system led to a decrease of both conversion and selectivity towards dimethyl succinate. Pulsed-field gradient (PFG)-NMR spectroscopy was used to assess the effect of water addition on the effective self-diffusivity of the reactant within the catalyst. NMR relaxation studies were also carried out to probe the strength of surface interaction of the reactant in the absence and presence of water. PFG-NMR studies revealed that the addition of water to the initial system, although increasing the dilution of the system, leads to a significant decrease of effective diffusion rate of the reactant within the catalyst. From T(1) and T(2) relaxation measurements it was possible to infer the strength of surface interaction of the reactant with the catalyst surface. The addition of water was found to inhibit the adsorption of the reactant over the catalyst surface, with the T(1)/T(2) ratio of 1,4-butanediol decreasing significantly when water was added. The results overall suggest that both the decrease of diffusion rate and adsorption strength of the reactant within the catalyst, due to water addition, limits the access of reactant molecules to the catalytic sites, which results in a decrease of reaction rate and conversion.

High-resolution structural insights into bone: a solid-state NMR relaxation study utilizing paramagnetic doping.

The hierarchical heterogeneous architecture of bone imposes significant challenges to structural and dynamic studies conducted by traditional biophysical techniques. High-resolution solid-state nuclear magnetic resonance (SSNMR) spectroscopy is capable of providing detailed atomic-level structural insights into such traditionally challenging materials. However, the relatively long data-collection time necessary to achieve a reliable signal-to-noise ratio (S/N) remains a major limitation for the widespread application of SSNMR on bone and related biomaterials. In this study, we attempt to overcome this limitation by employing the paramagnetic relaxation properties of copper(II) ions to shorten the (1)H intrinsic spin-lattice (T(1)) relaxation times measured in natural-abundance (13)C cross-polarization (CP) magic-angle-spinning (MAS) NMR experiments on bone tissues for the purpose of accelerating the data acquisition time in SSNMR. To this end, high-resolution solid-state (13)C CPMAS experiments were conducted on type I collagen (bovine tendon), bovine cortical bone, and demineralized bovine cortical bone, each in powdered form, to measure the (1)H T(1) values in the absence and in the presence of 30 mM Cu(II)(NH(4))(2)EDTA. Our results show that the (1)H T(1) values were successfully reduced by a factor of 2.2, 2.9, and 3.2 for bovine cortical bone, type I collagen, and demineralized bone, respectively, without reducing the spectral resolution and thus enabling faster data acquisition. In addition, paramagnetic quenching of particular (13)C NMR resonances on exposure to Cu(2+) ions in the absence of mineral was also observed, potentially suggesting the relative proximity of three main amino acids in the protein backbone (glycine, proline, and alanine) to the bone mineral surface.

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state.

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R 2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.

NMR and X-ray Study Revealing the Rigidity of Zeolitic Imidazolate Frameworks

NMR relaxation studies and spectroscopic measurements of zeolitic imidazolate framework-8 (ZIF-8) are reported. The dominant nuclear spin–lattice relaxation (T1) mechanism for ZIF-8 in air arises from atmospheric paramagnetic molecular oxygen. The 13C T1 measurements indicate that the oxygen interacts primarily with the imidazolate ring rather than the methyl substituent. Similar relaxation behavior was also observed in a ZIF with an unsubstituted ring, ZIF-4. Single-crystal X-ray diffraction was used to provide data for the study of the thermal ellipsoids of ZIF-8 at variable temperatures from 100 to 298 K, which further confirmed the rigid nature of this ZIF framework. These results highlight a rigid ZIF framework and are in contrast with dynamic metal–organic frameworks based on benzenedicarboxylate linking groups, for which the relaxation reflects the dynamics of the benzenedicarboxylate moiety.

NMR relaxation study of the effect of molecular weight on the topological structure and molecular mobility of linear polymers

NMR relaxation study of the effect of molecular weight on the topological structure and molecular mobility of linear polymers

Denaturation of HIV-1 Protease (PR) Monomer by Acetic Acid: Mechanistic and Trajectory Insights from Molecular Dynamics Simulations and NMR

Inside a living cell there can be a variety of interactions for any given protein, which serve to regulate denaturation and renaturation processes. Insights into some of them can be obtained by in vitro studies using various denaturing agents. In this study, all-atom MD simulations in explicit solvent and NMR relaxation studies were performed on HIV-1 Protease (PR) in 9 M acetic acid (AcOH) (the commonly used denaturant during PR preparation). Following previous reports that denaturation proceeds via dissociation of the dimer into monomers, unfolding of the monomer by acetic acid has been explicitly investigated here. Direct visualization of the denaturation process and evidence for the mechanism of denaturation have been presented. Our simulations reveal that the denaturation of the PR monomer is caused due to direct interaction between acetic acid molecules and PR. Autocorrelation of N-H vectors calculated from the simulations have revealed that the α-helix and the surrounding β-strands represent the sensitive regions of the PR that respond maximally to the change in the solvent environment around the PR and are prone to disruption by acetic acid. This disruption is caused due to increased penetration of the acetic acid molecules into the PR structure by formation of preferred tertiary contacts and hydrogen bonds between the PR and acetic acid molecules. Following the loss of these critical interactions, the PR follows a random and non-equilibrating path on the conformation landscape and cycles between different denatured extended and compact states.

Microstructure of cellulose: NMR relaxation study

The models of structural organization of cellulose are reviewed within the framework of the semi-crystalline concept. Special attention is given to two crystalline modifications of cellulose: Iα and Iβ. The results of low-resolution 1H NMR studies of the supramolecular structure of cellulose are considered. The mathematical model describing the line shape of the solid-state NMR spectrum of cellulose is presented. A scheme for the structure of cellulose microfibrils that fits most of the known model concepts is proposed. It is shown that this scheme can be used to determine the major supramolecular parameters of this polymer.

NMR relaxation dispersion of Miglyol molecules confined inside polymeric micro‐capsules

Frequency dependent NMR relaxation studies have been carried out on Miglyol molecules confined inside core shell polymeric capsules to obtain a correlation between capsule dimension and the measurable parameters. The polymeric capsules were prepared using an interfacial polymerization technique for three different concentrations of Miglyol. It was shown that the variation of Miglyol concentration influences the capsule dimension. Their average size was estimated using the pulsed field gradient diffusometry technique. The relaxation dispersion curves were obtained at room temperature by a combined use of a fast field cycling instrument and a high-field instrument. The frequency dependence of relaxation rate shows a transition from a diffusion-limited to a surface-limited relaxation regime.

NMR Relaxation Study of Cholesterol Binding with Plant Metabolites

Nuclear magnetic resonance relaxation technique has been applied to study the interaction of cholesterol and its oxidation products with plant metabolite, glycyrrhizic acid. In all cases, the decay kinetics of echo signal shows bi-exponential behavior. It was demonstrated that the fast component of the decay kinetics belongs to the complex with stoichiometry of 1:2. The stability constants and thermodynamic parameters of these complexes have been measured. We propose that the complexation with glycyrrhizic acid could be an effective approach to regulate the cholesterol level inside and outside of cage membranes. In addition, the importance of this approach as a tool for reducing the cytotoxicity of the oxidation products of cholesterol is under discussion.

NMR relaxation study of the phase transitions and relaxation mechanisms of the alums MCr(SO 4) 2·12H 2O ( M=Rb and Cs) single crystals

The physical properties and phase transition mechanisms of MCr(SO 4) 2·12H 2O ( M=Rb and Cs) single crystals have been investigated. The phase transition temperatures, NMR spectra, and the spin-lattice relaxation times T 1 of the 87Rb and 133Cs nuclei in the two crystals were determined using DSC and FT NMR spectroscopy. The resonance lines and relaxation times of the 87Rb and 133Cs nuclei undergo significant changes at the phase transition temperatures. The sudden changes in the splitting of the Rb and Cs resonance lines are attributed to changes in the local symmetry of their sites, and the changes in the temperature dependences of T 1 are related to variations in the symmetry of the octahedra of water molecules surrounding Rb + and Cs +. We also compared these 87Rb and 133Cs NMR results with those obtained for the trivalent cations Cr and Al in MCr(SO 4) 2·12H 2O and MAl(SO 4) 2·12H 2O crystals.

Comparison of In Vitro Metabolism of Ticlopidine by Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4

A recent X-ray crystal structure of a rabbit cytochrome P450 2B4 (CYP2B4)-ticlopidine complex indicated that the compound could be modeled with either the thiophene or chlorophenyl group oriented toward the heme prosthetic group. Subsequent NMR relaxation and molecular docking studies suggested that orientation with the chlorophenyl ring closer to the heme was the preferred one. To evaluate the predictive value of these findings, the oxidation of ticlopidine by reconstituted CYP2B4 was studied and compared with CYP2B6, in which the thiophene portion of the molecule likely orients toward the heme. In vitro incubation of ticlopidine with both enzymes yielded the same set of metabolites: 7-hydroxyticlopidine (M1), 2-oxoticlopidine (M2), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M3), 5-(2-chlorobenzyl)thieno[3,2-c]pyridin-5-ium metabolite (M4), ticlopidine N-oxide (M5), and ticlopidine S-oxide dimer, a dimerization product of ticlopidine S-oxide (M6). The rates of metabolite formation deviated markedly from linearity with time, consistent with the known inactivation of CYP2B6 by ticlopidine. Fitting to a first-order equation yielded similar rate constants (k(obs)) for both enzymes. However, the amplitude (R(max)) of M1 and M6 formation was 4 to 5 times higher for CYP2B6 than CYP2B4, indicating a greater residence time of ticlopidine with its thiophene ring closer to heme in CYP2B6. In contrast, CYP2B4 formed M4 and M5 in more abundance than CYP2B6, indicating an alternate orientation. Overall, the results suggest that the preferential orientation of ticlopidine in the active site of CYP2B4 predicted by X-ray crystallography and NMR studies is unproductive and that ticlopidine likely reorients within CYP2B4 to a more productive mode.

Modeling Conformational Ensembles of Slow Functional Motions in Pin1-WW

Protein-protein interactions are often mediated by flexible loops that experience conformational dynamics on the microsecond to millisecond time scales. NMR relaxation studies can map these dynamics. However, defining the network of inter-converting conformers that underlie the relaxation data remains generally challenging. Here, we combine NMR relaxation experiments with simulation to visualize networks of inter-converting conformers. We demonstrate our approach with the apo Pin1-WW domain, for which NMR has revealed conformational dynamics of a flexible loop in the millisecond range. We sample and cluster the free energy landscape using Markov State Models (MSM) with major and minor exchange states with high correlation with the NMR relaxation data and low NOE violations. These MSM are hierarchical ensembles of slowly interconverting, metastable macrostates and rapidly interconverting microstates. We found a low population state that consists primarily of holo-like conformations and is a “hub” visited by most pathways between macrostates. These results suggest that conformational equilibria between holo-like and alternative conformers pre-exist in the intrinsic dynamics of apo Pin1-WW. Analysis using MutInf, a mutual information method for quantifying correlated motions, reveals that WW dynamics not only play a role in substrate recognition, but also may help couple the substrate binding site on the WW domain to the one on the catalytic domain. Our work represents an important step towards building networks of inter-converting conformational states and is generally applicable.

The role of NMR in the study of partially ordered materials: Perspectives and challenges

The development of NMR techniques applied in the last 10 years to partially oriented systems, and in particular to liquid crystals (LCs), is the object of this brief perspective. The evolution of NMR methods (i.e., new NMR pulse sequences) and the improvement of both theoretical models and mathematic tools for the analysis of NMR data (specifically, for partially ordered systems) allowed scientists to extend their research to increasingly complex materials, such as dendrimers, polymers, and membranes, and to investigate unique phenomena, such as field-induced alignment and confining effects. Furthermore, the fast development of nanoscience and biomedicine is offering a rich variety of new “physical chemical” problems related to partially ordered materials. Starting from a brief perspective of recent works on thermotropic and lyotropic LCs based on different NMR methods, new challenges in this field will be drawn. Moreover, recent selected research works will be discussed in detail with particular emphasis on: (i) the effect of high magnetic fields on the supramolecular structure of chiral liquid-crystalline phases, such as the SmC*, TGBA*, and “de Vries”-type SmA* phases, by means of solid-state 2H NMR; (ii) the slow dynamics in the isotropic phase of bent-core LCs (BLCs) and of liquid single-crystal elastomers evidenced by 2H NMR relaxation studies; and (iii) the influence of the LC environment on the conformational properties of rod-like mesogens studied by high-resolution solid-state 13C NMR methods. This work aims to offer an occasion of reflection on this field of physical chemistry with a glance at future trends and challenges in view of the celebration of the International Year of Chemistry, 2011.