Nuclear Magnetic Resonance Spin Relaxation Research Articles

Structural Characterization of Food Allergens by Nuclear Magnetic Resonance Spectroscopy.

As allergies, especially those triggered by food, are becoming more and more prevalent, it is of increasing importance to fully understand the structures and dynamic behaviors of allergenic proteins along with their interactions with potential natural ligands. Therefore, we have established a solid routine to achieve structural characterization of food allergens, especially for birch pollen-related cross-reactive proteins from the class 10 of pathogenesis-related proteins (PR-10), by nuclear magnetic resonance (NMR) spectroscopy. Following expression of the desired allergen in Escherichia coli in isotope-labeled minimal media, the three-dimensional solution structures of these proteins can be determined, and insight into ligand binding mechanics and structural dynamic properties are accessible through NMR spin relaxation experiments.

Intracellular environment can change protein conformational dynamics in cells through weak interactions.

Conformational dynamics is important for protein functions, many of which are performed in cells. How the intracellular environment may affect protein conformational dynamics is largely unknown. Here, loop conformational dynamics is studied for a model protein in Escherichia coli cells by using nuclear magnetic resonance (NMR) spectroscopy. The weak interactions between the protein and surrounding macromolecules in cells hinder the protein rotational diffusion, which extends the dynamic detection timescale up to microseconds by the NMR spin relaxation method. The loop picosecond to microsecond dynamics is confirmed by nanoparticle-assisted spin relaxation and residual dipolar coupling methods. The loop interactions with the intracellular environment are perturbed through point mutation of the loop sequence. For the sequence of the protein that interacts stronger with surrounding macromolecules, the loop becomes more rigid in cells. In contrast, the mutational effect on the loop dynamics in vitro is small. This study provides direct evidence that the intracellular environment can modify protein loop conformational dynamics through weak interactions.

Conformational Dynamics of the Hepatitis B Virus Pre-genomic RNA on Multiple Time Scales: Implications for Viral Replication

Human hepatitis B virus (HBV) replication is initiated by the binding of the viral polymerase (P) to epsilon (ε), an ≈85-nucleotide (nt) cis-acting regulatory stem-loop RNA located at the 5′-end of the pre-genomic RNA (pgRNA). This interaction triggers P and pgRNA packaging and protein-primed reverse transcription and is therefore an attractive therapeutic target. Our recent nuclear magnetic resonance (NMR) structure of ε provides a useful starting point toward a detailed understanding of HBV replication, and hints at the functional importance of ε dynamics. Here, we present a detailed description of ε motions on the ps to ns and μs to ms time scales by NMR spin relaxation and relaxation dispersion, respectively. We also carried out molecular dynamics simulations to provide additional insight into ε conformational dynamics. These data outline a series of complex motions on multiple time scales within ε. Moreover, these motions occur in mostly conserved nucleotides from structural regions (i.e., priming loop, pseudo-triloop, and U43 bulge) that biochemical and mutational studies have shown to be essential for P binding, P-pgRNA packaging, protein-priming, and DNA synthesis. Taken together, our work implicates RNA dynamics as an integral feature that governs HBV replication.

Host-guest interactions and confinement effects in HZSM-5 and chabazite zeolites studied by low-field NMR spin relaxation

The characterization of fluid/solid interactions in porous materials is crucial for their design and optimization, most notably in applications such as adsorption and catalysis. Yet, probing interfacial phenomena of fluids confined in porous systems is particularly challenging. Nuclear magnetic resonance (NMR) spin relaxation has emerged in recent years as a rapid, non-invasive experimental technique to probe adsorbate/adsorbent interactions in mesoporous catalytic materials. More recently, NMR relaxation measurements performed on high-field (300 MHz) superconducting magnets have been successfully validated as a robust method to characterize acidity in HZSM-5 zeolites. Expanding such techniques in the context of low-field, bench-top NMR instruments would be highly beneficial as it would make NMR relaxation a much more appealing and accessible tool for non-invasive, rapid characterization of adsorbate/adsorbent interactions in zeolites and microporous materials. Herein, we validate the use of low-field, bench-top NMR spin relaxation as an indicator for characterizing host-guest interactions in microporous zeolitic materials, using water as guest molecules confined within two different zeolite frameworks, HZSM-5 and chabazite, with varying silica/alumina ratio. The results reported here demonstrate the robustness and sensitivity of low-field NMR relaxation measurements as a rapid screening tool for characterizing adsorption and molecular dynamics in microporous materials, with important implications for both academics and industrialists in terms of making the method more widely accessible, hence expanding the set of tools for materials chemistry and characterization.

NMR Spin Relaxation Theory of Biomolecules Undergoing Highly Asymmetric Exchange with Large Interaction Partners

The transient interactions of proteins and other molecules with much larger structures, such as synthetic or biological nanoparticles, lead to certain types of enhanced nuclear magnetic resonance (NMR) spin relaxation effects, which can be accurately measured by multidimensional solution NMR techniques. These relaxation effects provide new information about the nanostructures and the protein, their interactions, internal dynamics, and associated kinetic and thermodynamic parameters, such as exchange rates and populations. Although theoretical treatments exist that cover either the fast or slow exchange limits, a theoretical treatment that applies to all practically relevant exchange processes is still missing. A unified theoretical framework is presented for this purpose based on a stochastic Liouville equation (SLE). It covers nuclear spin dynamics, overall rotational diffusion of both the protein and the nanostructure, the exchange process between a free state and a bound state, and internal protein dynamics. Although the numerical implementation of the SLE typically involves large matrices, it is shown here that it is computationally still tractable for situations relevant in practice. Application of the theory demonstrates how transverse relaxation is substantially impacted by the kinetics of binding on a wide range of exchange timescales. It is further shown that when exchange occurs on the appropriate timescale, transverse relaxation is able to report on internal dynamics far slower than observable by traditional transverse relaxation experiments. The SLE will allow the realistic and quantitative interpretation of experimental NMR data reporting about transient protein-nanoparticle interactions, thereby providing a powerful tool for the characterization of protein dynamics modes on a vast range of timescales including motions that may be functionally relevant.

Reorientational dynamics of molecules in liquid methane: A molecular dynamics simulation study

Rotational motion of molecules in liquids plays an important role in determining the liquids' Nuclear Magnetic Resonance (NMR) spin-relaxation properties. The traditional theory of NMR spin relaxation assumes that the reorientational dynamics of molecules in liquids can be described by a continuous-time rotational-diffusion random walk with a single rotational-diffusion coefficient. However, recent experimental and theoretical studies have demonstrated that the simple rotational-diffusion model does not fully capture the rotational dynamics of molecules in liquids: for example, the reorientation of molecules in liquid water occurs through a combination of continuous-time rotational diffusion and discrete, large angular jumps resulting from collective rearrangements of the hydrogen-bond network of water.In order to obtain further insights into the origin of large angular jumps in the rotational motion of liquid water, we studied the rotational dynamics of methane - an apolar, non‑hydrogen-bonding liquid with a spherically symmetric molecule. The reorientational propagator and the ensemble-averaged Legendre polynomial reorientational functions of liquid methane were analysed using molecular dynamics simulations. On the femtosecond timescale, the reorientational motion of methane molecules was found to be characterised by free-rotation gas-like Gaussian decay of the reorientational Legendre polynomials, a result consistent with the available experimental data for liquid methane. On the long timescale (picoseconds) the decay of the reorientational Legendre polynomials was exponential, suggestive of a Debye-like continuous-time rotational diffusion behaviour. However, the ratios of the exponential time constants for different orders of the Legendre polynomials failed to match the rotational-diffusion model. We discuss the implications of these findings for the understanding of rotational motion of molecules in hydrogen-bonding and non‑hydrogen-bonding liquids.

Role of Backbone Dynamics in Modulating the Interactions of Disordered Ligands with the TAZ1 Domain of the CREB-Binding Protein.

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

Lysines in the RNA Polymerase II C-Terminal Domain Contribute to TAF15 Fibril Recruitment.

Many cancer-causing chromosomal translocations result in transactivating protein products encoding FET family (FUS, EWSR1, TAF15) low-complexity (LC) domains fused to a DNA binding domain from one of several transcription factors. Recent work demonstrates that higher-order assemblies of FET LC domains bind the carboxy-terminal domain of the large subunit of RNA polymerase II (RNA pol II CTD), suggesting FET oncoproteins may mediate aberrant transcriptional activation by recruiting RNA polymerase II to promoters of target genes. Here we use nuclear magnetic resonance (NMR) spectroscopy and hydrogel fluorescence microscopy localization and fluorescence recovery after photobleaching to visualize atomic details of a model of this process, interactions of RNA pol II CTD with high-molecular weight TAF15 LC assemblies. We report NMR resonance assignments of the intact degenerate repeat half of human RNA pol II CTD alone and verify its predominant intrinsic disorder by molecular simulation. By measuring NMR spin relaxation and dark-state exchange saturation transfer, we characterize the interaction of RNA pol II CTD with amyloid-like hydrogel fibrils of TAF15 and hnRNP A2 LC domains and observe that heptads far from the acidic C-terminal tail of RNA pol II CTD bind TAF15 fibrils most avidly. Mutation of CTD lysines in heptad position 7 to consensus serines reduced the overall level of TAF15 fibril binding, suggesting that electrostatic interactions contribute to complex formation. Conversely, mutations of position 7 asparagine residues and truncation of the acidic tail had little effect. Thus, weak, multivalent interactions between TAF15 fibrils and heptads throughout RNA pol II CTD collectively mediate complex formation.

Identification of Dynamic Modes in an Intrinsically Disordered Protein Using Temperature-Dependent NMR Relaxation.

The dynamic modes and time scales sampled by intrinsically disordered proteins (IDPs) define their function. Nuclear magnetic resonance (NMR) spin relaxation is probably the most powerful tool for investigating these motions delivering site-specific descriptions of conformational fluctuations from throughout the molecule. Despite the abundance of experimental measurement of relaxation in IDPs, the physical origin of the measured relaxation rates remains poorly understood. Here we measure an extensive range of auto- and cross-correlated spin relaxation rates at multiple magnetic field strengths on the C-terminal domain of the nucleoprotein of Sendai virus, over a large range of temperatures (268-298 K), and combine these data to describe the dynamic behavior of this archetypal IDP. An Arrhenius-type relationship is used to simultaneously analyze up to 61 relaxation rates per amino acid over the entire temperature range, allowing the measurement of local activation energies along the chain, and the assignment of physically distinct dynamic modes. Fast (τ ≤ 50 ps) components report on librational motions, a dominant mode occurs on time scales around 1 ns, apparently reporting on backbone sampling within Ramachandran substates, while a slower component (5-25 ns) reports on segmental dynamics dominated by the chain-like nature of the protein. Extending the study to three protein constructs of different lengths (59, 81, and 124 amino acids) substantiates the assignment of these contributions. The analysis is shown to be remarkably robust, accurately predicting a broad range of relaxation data measured at different magnetic field strengths and temperatures. The ability to delineate intrinsic modes and time scales from NMR spin relaxation will improve our understanding of the behavior and function of IDPs, adding a new and essential dimension to the description of this biologically important and ubiquitous class of proteins.

The Nedd4-1 WW Domain Recognizes the PY Motif Peptide through Coupled Folding and Binding Equilibria.

The four WW domains of human Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) interact with the PPxY (PY) motifs of the human epithelial Na(+) channel (hENaC) subunits, with the third WW domain (WW3*) showing the highest affinity. We have shown previously that the α-hENaC PY motif binding interface of WW3* undergoes conformational exchange on the millisecond time scale, indicating that conformational sampling plays a role in peptide recognition. To further understand this role, the structure and dynamics of hNedd4-1 WW3* were investigated. The nuclear Overhauser effect-derived structure of apo-WW3* resembles the domain in complex with the α-hENaC peptide, although particular side chain conformations change upon peptide binding, which was further investigated by molecular dynamics simulations. Model-free analysis of the (15)N nuclear magnetic resonance spin relaxation data showed that the apo and peptide-bound states of WW3* have similar backbone picosecond to nanosecond time scale dynamics. However, apo-WW3* exhibits pronounced chemical exchange on the millisecond time scale that is quenched upon peptide binding. (1)HN and (15)N Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments at various temperatures revealed that apo-WW3* exists in an equilibrium between the natively folded peptide binding-competent state and a random coil-like denatured state. The thermodynamics of the folding equilibrium was determined by fitting a thermal denaturation profile monitored by circular dichroism spectroscopy in combination with the CPMG data, leading to the conclusion that the unfolded state is populated to ∼ 20% at 37 °C. These results show that the binding of the hNedd4-1 WW3* domain to α-hENaC is coupled to the folding equilibrium.

IDPs: Less Disordered and More Ordered than Expected

In this issue of the Biophysical Journal, Solyom et al. (9) provide a telling example of structural preformation in intrinsically disordered proteins (IDPs) and the relevance of distinctly folded and compact conformational substates for molecular recognition processes.

Abnormal difference between the mobilities of left- and right-twisted conformations of C6H12N2 roto-symmetrical molecules at very low temperatures.

We report an abnormal difference of low-temperature mobility of left-twisted and right-twisted conformations of roto symmetric molecules C6H12N2 (dabco) located in the same positions in crystal Zn2(C8H4O4)2⋅C6H12N2. The difference between (1)H NMR (Nuclear Magnetic Resonance) spin-relaxation data for left-twisted and right-twisted molecules reaches ∼3 × 10(3) times at 8 K and tends to grow at lower temperatures. We argue that taking into account four-component relativistic Dirac wave functions in the vicinity of the nodal plane of dabco molecules and vacuum fluctuations due to virtual particle-antiparticle pairs can explain the changes which C6H12N2 conformations undergo at low temperatures.

TU-EF-BRA-01: NMR and Proton Density MRI of the 1D Patient

TU-EF-BRA-01: NMR and Proton Density MRI of the 1D Patient

TU‐EF‐BRA‐04: Into 2, 3, and 4 Dimensions

TU‐EF‐BRA‐04: Into 2, 3, and 4 Dimensions

TU-EF-BRA-03: Free Induction Decay (without the Decay) and Spin-Echo Imaging

TU-EF-BRA-03: Free Induction Decay (without the Decay) and Spin-Echo Imaging

TU-EF-BRA-02: Longitudinal Proton Spin Relaxation and T1-Imaging

TU-EF-BRA-02: Longitudinal Proton Spin Relaxation and T1-Imaging

TU-EF-BRA-00: MR Basics I

TU-EF-BRA-00: MR Basics I

Backbone and ILV methyl resonance assignments of E. coli thymidylate synthase bound to cofactor and a nucleotide analogue

Thymidylate synthase (TSase) is a 62 kDa homodimeric enzyme required for de novo synthesis of thymidine monophosphate in most organisms. This makes the enzyme an excellent target for anticancer and microbial antibiotic drugs. In addition, TSase has been shown to exhibit negative cooperativity and half-the-sites reactivity. For these collective reasons, TSase is widely studied, and much is known about its kinetics and structure as it progresses through a multi-step catalytic cycle. Recently, nuclear magnetic resonance spin relaxation has been instrumental in demonstrating the critical role of dynamics in enzyme function in small model systems. These studies raise questions about how dynamics affect function in larger enzymes with more complex reaction coordinates. TSase is an ideal candidate given its size, oligomeric state, cooperativity, and status as a drug target. Here, as a pre-requisite to spin relaxation studies, we present the backbone and ILV methyl resonance assignments of TSase from Escherichia coli bound to a substrate analogue and cofactor.

Probing Sequence-Specific DNA Flexibility in A-Tracts and Pyrimidine-Purine Steps by Nuclear Magnetic Resonance 13C Relaxation and Molecular Dynamics Simulations

Sequence-specific DNA flexibility plays a key role in a variety of cellular interactions that are critical for gene packaging, expression, and regulation, yet few studies have experimentally explored the sequence dependence of DNA dynamics that occur on biologically relevant time scales. Here, we use nuclear magnetic resonance (NMR) carbon spin relaxation combined with molecular dynamics (MD) simulations to examine the picosecond to nanosecond dynamics in a variety of dinucleotide steps as well as in varying length homopolymeric A(n)·T(n) repeats (A(n)-tracts, where n = 2, 4, or 6) that exhibit unusual structural and mechanical properties. We extend the NMR spin relaxation time scale sensitivity deeper into the nanosecond regime by using glycerol and a longer DNA duplex to slow overall tumbling. Our studies reveal a structurally unique A-tract core (for n > 3) that is uniformly rigid, flanked by junction steps that show increasing sugar flexibility with A-tract length. High sugar mobility is observed at pyrimidine residues at the A-tract junctions, which is encoded at the dinucleotide level (CA, TG, and CG steps) and increases with A-tract length. The MD simulations reproduce many of these trends, particularly the overall rigidity of A-tract base and sugar sites, and suggest that the sugar-backbone dynamics could involve transitions in sugar pucker and phosphate backbone BI ↔ BII equilibria. Our results reinforce an emerging view that sequence-specific DNA flexibility can be imprinted in dynamics occurring deep within the nanosecond time regime that is difficult to characterize experimentally at the atomic level. Such large-amplitude sequence-dependent backbone fluctuations might flag the genome for specific DNA recognition.

Interpretation of biomolecular NMR spin relaxation parametersThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Biomolecular nuclear magnetic resonance (NMR) spin relaxation experiments provide exquisite information on the picosecond to nanosecond timescale motions of bond vectors. Spin-lattice (T1) and spin-spin (T2) relaxation times and the steady-state nuclear Overhauser effect (NOE) are the first set of parameters extracted from typical 15N or 13C NMR relaxation experiments. Therefore, verifying that T1, T2, and NOE are consistent with theoretical predictions is an important step before carrying out the more detailed model-free and reduced spectral density mapping analyses commonly employed. In this mini-review, we discuss the essential motional parameters used to describe biomolecular dynamics in the context of a variety of examples of folded and intrinsically disordered proteins and peptides in aqueous and membrane mimetic environments. Estimates of these parameters can be used as input for an online interface, introduced herein, allowing plotting of trends of T1, T2, and NOE with magnetic field strength. The plots may serve as a first-check to the spectroscopist preparing to embark on a detailed NMR relaxation analysis.