NMR Relaxation Methods Research Articles

Mobility of sodium ions in agarose gels probed through combined single- and triple-quantum NMR

Metal ions, including biologically prevalent sodium ions, can modulate electrostatic interactions frequently involved in the stability of condensed compartments in cells. Quantitative characterization of heterogeneous ion dynamics inside biomolecular condensates demands new experimental approaches. Here we develop a 23Na NMR relaxation-based integrative approach to probe dynamics of sodium ions inside agarose gels as a model system. We exploit the electric quadrupole moment of spin-3/2 23Na nuclei and, through combination of single-quantum and triple-quantum-filtered 23Na NMR relaxation methods, disentangle the relaxation contribution of different populations of sodium ions inside gels. Three populations of sodium ions are identified: a population with bi-exponential relaxation representing ions within the slow motion regime and two populations with mono-exponential relaxation but at different rates. Our study demonstrates the dynamical heterogeneity of sodium ions inside agarose gels and presents a new experimental approach for monitoring dynamics of sodium and other spin-3/2 ions (e.g. chloride) in condensed environments.

Investigating the behaviour of NaCl brines and hydrocarbons in porous alumina using low-field NMR relaxation and diffusion methods.

The behaviour of multiple fluid phases within a porous medium is hard to predict. NMR measurements offer an excellent tool to probe such systems in a fast and non-invasive way. Such systems can be relevant to hydrocarbon recovery, catalysis, and CO2 and H2 geo-storage, among others. Since electrolyte solutions are always present in subsurface reservoirs, understanding their behaviour within porous media is highly important. In this study, we use NMR relaxation and diffusion methods to investigate the diffusion coefficients and strength of interactions between alumina surfaces and brines at various NaCl concentrations, focusing on the effect of salt concentration on transport and interactions within the porous structure. Furthermore, we study the spontaneous displacement of dodecane, a model hydrocarbon, from the same alumina pellets using the same brine solutions. Results show that brines of lower salinity consistently displace more dodecane in total, after soaking dodecane-saturated pellets in a brine solution for several days. This indicates that increased salt concentrations can reduce wettability towards the aqueous phase in simple metal oxide surfaces and highlights the capabilities of NMR to efficiently study such systems.

Probing diffusional exchange in mesoporous zeolite by NMR diffusion and relaxation methods

We propose NMR relaxation techniques to evaluate diffusional exchange between the different porosity compartments of heterogeneous catalyst extrudates in addition to NMR diffusion measurements. The two systems considered are close to industrial products and contain a wide distribution of pore sizes spanning from microporosity (2 types of zeolites CBV400 and CBV720), mesoporosity induced by the alumina binder, and macroporosity introduced during the shaping procedure. The question is to determine in which system the meso and macroporosity provide the best access to microporosity. Beside standard techniques, we measured the pore size distribution using NMR cryoporometry in the range 2 nm up to 1 μm, and the amount of microporosity below 2 nm from relaxation data at −29 °C. These measurements provide reference values independently of the connectivity or the hierarchical organization of the pore network. Long range diffusion was measured using methane and cyclohexane to evaluate the effect of macropores. When saturated with squalane, the diffusive exchange between micro and mesoporosity was evaluated using two techniques: (i) the comparison of the apparent mesoporosity fraction in the T2 distribution to the true value measured by cryoporometry, (ii) the measurement of exchange times using T2-exchange-T2 experiments. These two approaches give coherent results. The diffusive coupling was also observed with 2-propanol saturated extrudates, for which the surface residence time τS was evaluated from the interpretation of the 3 relaxation times T2, T1 and T1ρ; for the 2 samples τS differ by 2 orders of magnitude due to the very different Si/Al ratio of the 2 zeolites used in the extrudates. All these methods lead to the conclusion that the presence of mesoporosity in zeolite crystals is beneficial to the diffusive transport with the surrounding porosity only when molecule/zeolite interactions are not predominant.

Pressure, motion, and conformational entropy in molecular recognition by proteins

The thermodynamics of molecular recognition by proteins is a central determinant of complex biochemistry. For over a half-century, detailed cryogenic structures have provided deep insight into the energetic contributions to ligand binding by proteins. More recently, a dynamical proxy based on NMR-relaxation methods has revealed an unexpected richness in the contributions of conformational entropy to the thermodynamics of ligand binding. Here, we report the pressure dependence of fast internal motion within the ribonuclease barnase and its complex with the protein barstar. In what we believe is a first example, we find that protein dynamics are conserved along the pressure-binding thermodynamic cycle. The femtomolar affinity of the barnase-barstar complex exists despite a penalty by −TΔSconf of +11.7 kJ/mol at ambient pressure. At high pressure, however, the overall change in side-chain dynamics is zero, and binding occurs with no conformational entropy penalty, suggesting an important role of conformational dynamics in the adaptation of protein function to extreme environments. Distinctive clustering of the pressure sensitivity is observed in response to both pressure and binding, indicating the presence of conformational heterogeneity involving less efficiently packed alternative conformation(s). The structural segregation of dynamics observed in barnase is striking and shows how changes in both the magnitude and the sign of regional contributions of conformational entropy to the thermodynamics of protein function are possible.

Optimized radio frequency coil for noninvasive magnetic resonance relaxation detection of human finger

Noninvasive NMR measurement of human tissues, such as fingers, to achieve early detection for metabolic diseases is of important significance. The NMR relaxation measurements have a wide application prospect due to simplicity, portability, and low cost, as the static magnetic field is not required to be highly homogeneous. However, the inhomogeneous radiofrequency (RF) magnetic field (B1) results in errors in the magnetic resonance relaxation times. This is inevitable in in-vivo localized human tissue measurements with a portable MR scanner, as signals from tissues close to the edge of RF coil are excited with a different B1 field amplitude. A novel RF coil termed T coil with high B1 field homogeneity is presented. Numerical simulation and phantom measurements were implemented. The novel RF coil was compared with a regular solenoid coil and a variable width coil. In-vivo experiments were performed. The T coil has a better B1 field homogeneity than the regular solenoid coil and the variable width coil, producing more accurate magnetic resonance relaxation times. Improved detection accuracy has been achieved with the T coil. This work may promote the development of noninvasive human tissue diagnosis based on NMR relaxation methods.

Observation of Sub-Microsecond Protein Methyl-Side Chain Dynamics by Nanoparticle-Assisted NMR Spin Relaxation.

Amino-acid side-chain properties in proteins are key determinants of protein function. NMR spin relaxation of side chains is an important source of information about local protein dynamics and flexibility. However, traditional solution NMR relaxation methods are most sensitive to sub-nanosecond dynamics lacking information on slower ns-μs time-scale motions. Nanoparticle-assisted NMR spin relaxation (NASR) of methyl-side chains is introduced here as a window into these ns-μs dynamics. NASR utilizes the transient and nonspecific interactions between folded proteins and slowly tumbling spherical nanoparticles (NPs), whereby the increase of the relaxation rates reflects motions on time scales from ps all the way to the overall tumbling correlation time of the NPs ranging from hundreds of ns to μs. The observed motional amplitude of each methyl group can then be expressed by a model-free NASR S2 order parameter. The method is demonstrated for 2H-relaxation of CH2D methyl moieties and cross-correlated relaxation of CH3 groups for proteins Im7 and ubiquitin in the presence of anionic silica-nanoparticles. Both types of relaxation experiments, dominated by either quadrupolar or dipolar interactions, yield highly consistent results. Im7 shows additional dynamics on the intermediate time scales taking place in a functionally important loop, whereas ubiquitin visits the majority of its conformational substates on the sub-ns time scale. These experimental observations are in good agreement with 4-10 μs all-atom molecular dynamics trajectories. NASR probes side-chain dynamics on a much wider range of motional time scales than previously possible, thereby providing new insights into the interplay between protein structure, dynamics, and molecular interactions that govern protein function.

Rapid measurement of heteronuclear transverse relaxation rates using non-uniformly sampled R1ρ accordion experiments.

Multidimensional, heteronuclear NMR relaxation methods are used extensively to characterize the dynamics of biological macromolecules. Acquisition of relaxation datasets on proteins typically requires significant measurement time, often several days. Accordion spectroscopy offers a powerful means to shorten relaxation rate measurements by encoding the "relaxation dimension" into the indirect evolution period in multidimensional experiments. Time savings can also be achieved by non-uniform sampling (NUS) of multidimensional NMR data, which is used increasingly to improve spectral resolution or increase sensitivity per unit time. However, NUS is not commonly implemented in relaxation experiments, because most reconstruction algorithms are inherently nonlinear, leading to problems when estimating signal intensities, relaxation rate constants and their error bounds. We have previously shown how to avoid these shortcomings by combining accordion spectroscopy with NUS, followed by data reconstruction using sparse exponential mode analysis, thereby achieving a dramatic decrease in the total length of longitudinal relaxation experiments. Here, we present the corresponding transverse relaxation experiment, taking into account the special considerations required for its successful implementation in the framework of the accordion-NUS approach. We attain the highest possible precision in the relaxation rate constants by optimizing the NUS scheme with respect to the Cramér-Rao lower bound of the variance of the estimated parameter, given the total number of sampling points and the spectrum-specific signal characteristics. The resulting accordion-NUS relaxation experiment achieves comparable precision in the parameter estimates compared to conventional CPMG (Carr-Purcell-Meiboom-Gill) or spin-lock experiments while saving an order of magnitude in experiment time.

Sensitivity of Proton NMR Relaxation and Proton NMR Diffusion Measurements to Olive Oil Adulterations with Vegetable Oils.

Olive oils and, in particular, extra-virgin olive oils (EVOOs) are one of the most frauded food. Among the different adulterations of EVOOs, the mixture of high-quality olive oils with vegetable oils is one of the most common in the market. The need for fast and cheap techniques able to detect extra-virgin olive oil adulterations was the main motivation for the present research work based on 1H NMR relaxation and diffusion measurements. In particular, the 1H NMR relaxation times, T1 and T2, measured at 2 and 100 MHz on about 60 EVOO samples produced in Italy are compared with those measured on four different vegetable oils, produced from macadamia nuts, linseeds, sunflower seeds, and soybeans. Self-diffusion coefficients on this set of olive oils and vegetable oil samples were measured by means of the 1H NMR diffusion ordered spectroscopy (DOSY) technique, showing that, except for the macadamia oil, other vegetable oils are characterized by an average diffusion coefficient sensibly different from extra-virgin olive oils. Preliminary tests based on both NMR relaxation and diffusometry methods indicate that eventual adulterations of EVOO with linseed oil and macadamia oil are the easiest and the most difficult frauds to be detected, respectively.

Backbone Dynamics of the TAZ1 Domain of the Creb-Binding Protein Modulate Competition Between Disordered Ligands

Intrinsically disordered proteins (IDPs) play important roles in a multitude of cellular processes; however, the mechanisms by which disordered proteins carry out their diverse roles as modulators of cellular signaling are not yet completely understood. IDPs utilize a wide range of mechanisms to interact with folded proteins within the cell, and in many cases, must compete for binding of protein interaction partners. Yet, little is known about the contributions of the folded proteins to the binding and specificity of interactions with multiple disordered ligands. Are folded protein interaction partners simply scaffolds for IDP binding or do they undergo structural and dynamic changes to accommodate different disordered ligands? Using the folded TAZ1 domain of the transcriptional coactivator CBP and the disordered proteins HIF-1α and CITED2 as an example, we demonstrate that conformational dynamics and structural changes of TAZ1 are critical for binding and exchange of different IDPs. CITED2 competes with HIF-1α for TAZ1 binding through an extremely efficient unidirectional allosteric switch that relies not only on the physicochemical properties of the disordered proteins but also on the structure and dynamics of TAZ1. To gain further mechanistic insight into this process, we used NMR relaxation methods to obtain a detailed description of the backbone dynamics that contribute to TAZ1 binding and competition. We found that TAZ1 displays altered backbone dynamics in its free and bound states, with changes in TAZ1 dynamics observed not only in the HIF-1α and CITED2 binding sites but also in regions that differ in structure when bound to HIF-1α or CITED2. These data suggest that backbone dynamics in TAZ1 play a role in modulating binding of disordered ligands and highlight the importance of characterizing both folded and disordered partners in molecular interactions.

Collagen I Weakly Interacts with the β-Sheets of β2-Microglobulin and Enhances Conformational Exchange To Induce Amyloid Formation.

Amyloidogenesis is significant in both protein function and pathology. Amyloid formation of folded, globular proteins is commonly initiated by partial or complete unfolding. However, how this unfolding event is triggered for proteins that are otherwise stable in their native environments is not well understood. The accumulation of the immunoglobulin protein β2-microglobulin (β2m) into amyloid plaques in the joints of long-term hemodialysis patients is the hallmark of dialysis-related amyloidosis (DRA). While β2m does not form amyloid unassisted near neutral pH in vitro, the localization of β2m deposits to joint spaces suggests a role for the local extracellular matrix (ECM) proteins, specifically collagens, in promoting amyloid formation. Indeed, collagen and other ECM components have been observed to facilitate β2m amyloid formation, but the large size and anisotropy of the complex, combined with the low affinity of these interactions, have limited atomic-level elucidation of the amyloid-promoting mechanism(s) by these molecules. Using solution NMR approaches that uniquely probe weak interactions in large molecular weight complexes, we are able to map the binding interfaces on β2m for collagen I and detect collagen I-induced μs–ms time-scale dynamics in the β2m backbone. By combining solution NMR relaxation methods and 15N-dark-state exchange saturation transfer experiments, we propose a model in which weak, multimodal collagen I−β2m interactions promote exchange with a minor population of amyloid-competent species to induce fibrillogenesis. The results portray the intimate role of the environment in switching an innocuous protein into an amyloid-competent state, rationalizing the localization of amyloid deposits in DRA.

Crystallinity of polyolefins with large side groups by low-field 1H NMR T2 relaxometry: Isotactic Polybutene-1 with form II and I crystals

Phase composition and molecular mobility were studied using 1H NMR T2 relaxometry in isotactic polybutene-1 (iPB-1) with two polymorphs - form I and II crystals. Several types of NMR relaxation methods and data analysis were evaluated for determining the most reliable way for studying physical phases in iPB-1. Three-phase model provided the most appropriate description of the phase composition in iPB-1, i.e., a crystal-amorphous interface separates the crystalline and the amorphous phases. Due to complex molecular mobility in iPB-1, the amount of rigid fraction should be considered as NMR crystallinity number. Two types of chain segments are present in the amorphous phase: (1) chain segments with anisotropic mobility due to chain anchoring to crystals and chain entanglements; and (2) highly mobile chain end segments. The polymorphic phase II to I transition causes significant immobilization of polymer chains in the crystalline and the amorphous phases. Molecular weight of iPB-1 largely influences phase composition and molecular mobility in crystalline and amorphous phases.

The Nature of the Supramolecular Structural Variation and Hydrophilic Properties of Cellulose during Water Sorption

Water sorption on cellulose was studied in detail. The mechanism of the adsorption–desoprtion hysteresis loop was considered using NMR relaxation methods and sorption phenomena. The degree of crystallinity was found to decrease in cellulose during water sorption, while the specific surface area of cellulose increased because a wedging pressure arose as a new adsorbent–adsorbate interface formed. The most significant changes in these parameters were observed during desorption and were accompanied by the formation of an additional capillary-porous system in cellulose. Correlations were observed between the surface tension coefficient at the solid–liquid interface, the adsorption equilibrium constant, and the net heat of adsorption.

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR.

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

Ordering effect of protein surfaces on water dynamics: NMR relaxation study

Proteins in solution affect the structural and dynamic properties of the bulk water at the protein-water interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step toward the determination of the size of the water ordering contributions originate in proteins systems.

Mesoscopic Dynamics in Phospholipid Membranes under Osmotic Stress

Phospholipid membranes are biological liquid crystals exhibiting long-range molecular order [1]. Intermolecular interactions at mesoscopic length scales are key for explaining lipid-protein interactions and biomembrane functioning. Elastic deformations in such materials are manifested as director fluctuations with timescales spanning picoseconds to seconds [2]. Here we examine effects of osmotic stress on liquid-crystalline properties of lipid membranes using NMR relaxation methods. Membrane osmotic stress was monitored by addition of osmolytes and gravimetric dehydration. Solid-state 2H longitudinal (R1Z) and transverse quadrupolar-echo decay (R2QE) rates were measured for acyl-chain-perdeuterated lipid multilamellar dispersions. Plots of R1Z rates versus squared segmental order parameters (SCD2) follow an empirical linear function (square-law) suggesting the emergence of 3-D director fluctuations [2]. Slopes of these plots are sensitive to osmotic stress and invariant under temperature variation. Linear trends were also observed for R2QE rates but limited to segments deeper in the bilayer. Enhanced R2QE rates and their temperature dependence indicate additional relaxation contributions from slower dynamics. The dependence of R2QEon SCD2 near the headgroup suggests the onset of 2-D surface undulations. Membrane composition at the transition of 3-D to 2-D collective fluctuations has important implications for biomembrane functioning. The observed slow dynamics are due to mesoscopic phenomena that explain bulk material properties, where the connection between mechanical properties and NMR relaxation involves the fluctuation dissipation theorem. Restricted water accessibility into bilayer may suppress slow dynamic modes at high osmotic stress. Analogous studies with cholesterol in raft-like lipid mixtures [3] report on membrane compositions relevant for biomembrane function. Osmotic stress-mediated lipid dynamics thus opens a new window to exploring their coupling to biomembrane function. [1] T.R. Molugu et al. (2017) Chem. Rev. 117, 12087. [2] Leftin, A. et al. (2011) BBA 1808, 818. [3] T.R. Molugu et al. (2016) Chem. Phys. Lipids 199, 39.

Protein Dynamics revealed by NMR Relaxation Methods.

Structural biology often focuses primarily on three-dimensional structures of biological macromolecules, deposited in the Protein Data Bank (PDB). This resource is a remarkable entity for the world-wide scientific and medical communities, as well as the general public, as it is a growing translation into three-dimensional space of the vast information in genomic databases, e.g. GENBANK. There is, however, significantly more to understanding biological function than the three-dimensional coordinate space for ground-state structures of biomolecules. The vast array of biomolecules experiences natural dynamics, interconversion between multiple conformational states, and molecular recognition and allosteric events that play out on timescales ranging from picoseconds to seconds. This wide range of timescales demands ingenious and sophisticated experimental tools to sample and interpret these motions, thus enabling clearer insight into functional annotation of the PDB. NMR spectroscopy is unique in its ability to sample this range of timescales at atomic resolution and in physiologically relevant conditions using spin relaxation methods. The field is constantly expanding to provide new creative experiments, to yield more detailed coverage of timescales, and to broaden the power of interpretation and analysis methods. This review highlights the current state of the methodology and examines the extension of analysis tools for more complex experiments and dynamic models. The future for understanding protein dynamics is bright, and these extended tools bring greater compatibility with developments in computational molecular dynamics, all of which will further our understanding of biological molecular functions. These facets place NMR as a key component in integrated structural biology.

Dynamics of dehaloperoxidase-hemoglobin A derived from NMR relaxation spectroscopy and molecular dynamics simulation

Dehaloperoxidase-hemoglobin is the first hemoglobin identified with biologically-relevant oxidative functions, which include peroxidase, peroxygenase and oxidase activities. Herein we report a study of the protein backbone dynamics of DHP using heteronuclear NMR relaxation methods and molecular dynamics (MD) simulations to address the role of protein dynamics in switching from one function to another. The results show that DHP's backbone helical regions and turns have average order parameters of S2 = 0.87 ± 0.03 and S2 = 0.76 ± 0.08, respectively. Furthermore, DHP is primarily a monomer in solution based on the overall tumbling correlation time τm is 9.49 ± 1.65 ns calculated using the prolate diffusion tensor model in the program relax. A number of amino acid residues have significant Rex using the Lipari-Szabo model-free formalism. These include Lys3, Ile6, Leu13, Gln18, Arg32, Ser48, Met49, Thr56, Phe60, Arg69, Thr71 Cys73, Ala77, Asn81, Gly95, Arg109, Phe115, Leu127 and Met136, which may experience slow conformational motions on the microseconds-milliseconds time scale according to the model. Caution should be used when the model contains >4 fitting parameters. The program caver3.0 was used to identify tunnels inside DHP obtained from MD simulation snapshots that are consistent with the importance of the Xe binding site, which is located at the central intersection of the tunnels. These tunnels provide diffusion pathways for small ligands such as O2, H2O and H2O2 to enter the distal pocket independently of the trajectory of substrates and inhibitors, both of which are aromatic molecules.

Framework vs. side-chain amphidynamic behaviour in oligo-(ethylene oxide) functionalised covalent-organic frameworks.

We present a family of covalent organic frameworks that have been functionalized with oligo-(ethylene oxide) chains of varying lengths. Because of the open structure of the COFs, the side chains do not interfere with their crystallization obtaining materials with predictable crystal structure. The difference in length of the side-chains allowed for the determination of amphidynamic behaviour with the use of 13C solid-state NMR relaxation methods. Computational calculations further contribute to understanding the atomistic dynamic behaviour of the different atoms. This study demonstrates the ability to design complex behaviour in organic crystals.

Hydration of copper(II) amino acids complexes.

Hydration of the copper(II) bis-complexes with glycine, serine, lysine, and aspartic acid was studied by DFT and MD simulation methods. The distances between copper(II) and water molecules in the 1st and 2nd coordination shells, the average number of water molecules and their mean residence times in the hydration shells were calculated. Good agreement was observed between the values obtained and those found by DFT and NMR relaxation methods. Influence of the functional groups of the ligands and the cis-trans isomerism of the complexes on the structural and dynamical parameters of the hydration shells was displayed and explained. Analysis of the MD trajectories reveals the competition for a copper(II) axial position between water molecules or water molecules and the functional chain groups of the ligands and confirms the suggestion on the pentacoordination of copper(II) in such complexes. MD simulations show that only one axial position of Cu(II) is basically occupied at each time step while in average the coordination number more than 5 is observed. © 2017 Wiley Periodicals, Inc.

Increased Affinity of Small Gold Particles for Glycerol Oxidation over Au/TiO2 Probed by NMR Relaxation Methods

The aerobic oxidation of glycerol in aqueous solution over Au/TiO2 catalysts has been studied, and the effect of Au loading by wet impregnation, in the range 0.5–5% Au, has been assessed. Low metal loading favors the deposition of smaller particles, whereas higher loadings lead to the formation of much larger gold particles, as revealed by scanning transmission electron microscopy (STEM) analysis. Reaction studies show that a higher metal loading has a detrimental effect on the catalyst activity, which decreases significantly as the Au load increases. In addition to reaction studies, 1H NMR T1/T2 relaxation time measurements have been used to assess the effect of metal loading and particle size on the adsorption properties of glycerol (reactant) and water (solvent) within the catalyst. The NMR results show that the adsorption properties of glycerol relative to water as a function of the Au loading have a similar trend to that observed for the reactivity, with glycerol exhibiting a higher surface affinity ...