NMR Sample Research Articles

Conserved Surface Features Form the Double-stranded RNA Binding Site of Non-structural Protein 1 (NS1) from Influenza A and B Viruses

Influenza A viruses cause a highly contagious respiratory disease in humans and are responsible for periodic widespread epidemics with high mortality rates. The influenza A virus NS1 protein (NS1A) plays a key role in countering host antiviral defense and in virulence. The 73-residue N-terminal domain of NS1A (NS1A-(1-73)) forms a symmetric homodimer with a unique six-helical chain fold. It binds canonical A-form double-stranded RNA (dsRNA). Mutational inactivation of this dsRNA binding activity of NS1A highly attenuates virus replication. Here, we have characterized the unique structural features of the dsRNA binding surface of NS1A-(1-73) using NMR methods and describe the 2.1-A x-ray crystal structure of the corresponding dsRNA binding domain from human influenza B virus NS1B-(15-93). These results identify conserved dsRNA binding surfaces on both NS1A-(1-73) and NS1B-(15-93) that are very different from those indicated in earlier "working models" of the complex between dsRNA and NS1A-(1-73). The combined NMR and crystallographic data reveal highly conserved surface tracks of basic and hydrophilic residues that interact with dsRNA. These tracks are structurally complementary to the polyphosphate backbone conformation of A-form dsRNA and run at an approximately 45 degrees angle relative to the axes of helices alpha2/alpha2'. At the center of this dsRNA binding epitope, and common to NS1 proteins from influenza A and B viruses, is a deep pocket that includes both hydrophilic and hydrophobic amino acids. This pocket provides a target on the surface of the NS1 protein that is potentially suitable for the development of antiviral drugs targeting both influenza A and B viruses.

Real-time imaging of the spatial distribution of rf-heating in NMR samples during broadband decoupling

Continuous radio-frequency (rf) irradiation during decoupling and spin-lock periods in NMR pulse sequences may lead to undesired sample heating. Heat-sensitive samples can suffer damage from the sudden temperature rise which cannot be adequately compensated by the temperature control system. Moreover, as the heating is spatially inhomogeneous, higher temperature increases can arise locally than are indicated by the average increase detected by the temperature controller. In this work we present a technique that allows measurement of a real-time 2D-image of the temperature distribution inside an NMR sample during an experiment involving rf-heating. NMR imaging methods have previously been used to project the temperature distribution inside an NMR sample onto a single spatial axis or to acquire steady-state 2D- temperature distributions. The real-time 2D-temperature profiles obtained with our procedure provide much more detailed data. Our results show, that not only inhomogeneous heating but also inhomogeneous sample cooling contribute to the build-up of temperature gradients across the sample. The technique can be used to visualize rf-heating in order to protect sensitive samples and to experimentally test new coil geometries or to guide probehead design.

NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

Trans-4-tert-Butyl-1-methylcyclohexanol hemihydrate

The title compound, C11H22O·0.5H2O, is a hemihydrate of trans-4-tert-butyl-1-methyl­cyclo­hexa­nol, containing one water and two organic mol­ecules in the asymmetric unit. Crystals were obtained from an NMR sample by very slow evaporation of the solvent. In the solid state, the title compound forms a double-layered structure with the organic and water mol­ecules connected by O—H⋯O hydrogen bonds.

Development of an NMR Interface Microchip “MICCS” for Direct Detection of Reaction Products and Intermediates of Micro-syntheses Using a “MICCS-NMR”

The development of an NMR interface microchip and its applications to the real-time monitoring of chemical reactions are described. The microchip device was named "MICCS" (MIcro Channeled Cell for Synthesis monitoring), and the method using it was named "MICCS-NMR". MICCS was inserted into a 5 mm Phi NMR sample tube. Thus standard solution NMR probes without any modifications can be used in MICCS-NMR measurements. A gap between MICCS and the sample tube was filled with a deuterated solvent for an NMR lock. The reaction temperature and reaction time in MICCS can be easily changed by adjusting the temperature of the NMR probe and changing the flow rates, respectively. The effectiveness of the MICCS-NMR was verified in the real-time monitoring of the Wittig reaction. Preliminary data on the direct detection of intermediates of the Grignard reaction is also reported. Besides real-time monitoring of chemical reactions, MICCS-NMR would be useful as a qualitative detection method for microchip-based synthesis.

Pivotal Steps Towards Quantification of Molecular Diffusion Coefficients by NMR

Using nuclear magnetic resonance (NMR) spectroscopy with a pair of pulsed field gradients (PFGs), Stajeskal and Tanner successfully measured molecular diffusion coefficients in solution in 1965. This method has since been used extensively in various applications, especially after the PFG was implemented in commercial NMR probes. Due to the nonuniformity of the PFG and radio frequency (RF) fields, molecules distributed throughout the sample experience different PFG and RF fields and contribute unevenly to the measured diffusion coefficients, resulting in considerable errors in conventional NMR diffusion experiments. By selective excitation of a central sample region with an offset-independent adiabatic inversion pulse and a PFG, a uniform RF field can be assumed, and the PFG can be represented as a linear approximation. Under these conditions, the molecules diffuse as if they were all experiencing the same effective gradient g(e), leading to a Gaussian signal decay as a function of the PFG strength. Quantitative measurement of molecular diffusion coefficients is therefore made possible. From the diffusion coefficient of a 90 % H(2)O/10 % D(2)O sample, it is convenient to calibrate g(e) with a Java program. In a similar way the nonlinearity of the PFG can be corrected.

Analysis of glycans in glycoproteins by diffusion-ordered nuclear magnetic resonance spectroscopy

Enzymatically cleaved glycans from sub-milligram quantities of erythropoietin (EPO) and ovalbumin have been analyzed, without further purification, by two-dimensional diffusion-ordered nuclear magnetic resonance spectroscopy. At NMR sample concentrations below 50 micromol L-1 the major components of the oligosaccharide fractions could be distinguished by their anomeric proton chemical shift and their size-dependent diffusion coefficients.

All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples

Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems.

Cores and pH-dependent Dynamics of Ferredoxin-NADP+ Reductase Revealed by Hydrogen/Deuterium Exchange

NMR-detected hydrogen/deuterium (H/D) exchange of amide protons is a powerful way for investigating the residue-based conformational stability and dynamics of proteins in solution. Maize ferredoxin-NADP(+) reductase (FNR) is a relatively large protein with 314 amino acid residues, consisting of flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADP(+))-binding domains. To address the structural stability and dynamics of FNR, H/D exchange of amide protons was performed using heteronuclear NMR at pD(r) values 8.0 and 6.0, physiologically relevant conditions mimicking inside of chloroplasts. At both pD(r) values, the exchange rate varied widely depending on the residues. The profiles of protected residues revealed that the highly protected regions matched well with the hydrophobic cores suggested from the crystal structure, and that the NADP(+)-binding domain can be divided into two subdomains. The global stability of FNR obtained by H/D exchange with NMR was higher than that by chemical denaturation, indicating that H/D exchange is especially useful for analyzing the residue-based conformational stability of large proteins, for which global unfolding is mostly irreversible. Interestingly, more dynamic conformation of the C-terminal subdomain of the NADP(+)-binding domain at pD(r) 8.0, the daytime pH in chloroplasts, than at pD(r) 6.0 is likely to be involved in the increased binding of NADP(+) for elevating the activity of FNR. In light of photosynthesis, the present study provides the first structure-based relationship of dynamics with function for the FNR-type family in solution.

A Model for Agonism and Antagonism in an Ancient and Ubiquitous cAMP-binding Domain

The cAMP-binding domain (CBD) is an ancient and conserved regulatory motif that allosterically modulates the function of a group of diverse proteins, thereby translating the cAMP signal into a controlled biological response. The main receptor for cAMP in mammals is the ubiquitous regulatory (R) subunit of protein kinase A. Despite the recognized significant potential for pharmacological applications of CBDs, currently only one group of competitive inhibitor antagonists is known: the (R(p))-cAMPS family of phosphorothioate cAMP analogs, in which the equatorial exocyclic oxygen of cAMP is replaced by sulfur. It is also known that the diastereoisomer (S(p))-cAMPS with opposite phosphorous chirality is a cAMP agonist, but the molecular mechanism of action of these analogs is currently not fully understood. Previous crystallographic and unfolding investigations point to the enhanced CBD dynamics as a key determinant of antagonism. Here, we investigate the (R(p))- and (S(p))-cAMPS-bound states of R(CBD-A) using a comparative NMR approach that reveals a clear chemical shift and dynamic NMR signature, differentiating the (S(p))-cAMPS agonist from the (R(p))-cAMPS antagonist. Based on these data, we have proposed a model for the (R(p)/S(p))-cAMPS antagonism and agonism in terms of steric and electronic effects on two main allosteric relay sites, Ile(163) and Asp(170), respectively, affecting the stability of a ternary inhibitory complex formed by the effector ligand, the regulatory and the catalytic subunits of protein kinase A. The proposed model not only rationalizes the existing data on the phosphorothioate analogs, but it will also facilitate the design of novel cAMP antagonists and agonists.

Structural Basis for the Interaction of the Myosin Light Chain Mlc1p with the Myosin V Myo2p IQ Motifs

Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.

Using low-E resonators to reduce RF heating in biological samples for static solid-state NMR up to 900 MHz

RF heating of solid-state biological samples is known to be a destabilizing factor in high-field NMR experiments that shortens the sample lifetime by continuous dehydration during the high-power cross-polarization and decoupling pulses. In this work, we describe specially designed, large volume, low-E 15N– 1H solid-state NMR probes developed for 600 and 900 MHz PISEMA studies of dilute membrane proteins oriented in hydrated and dielectrically lossy lipid bilayers. The probes use an orthogonal coil design in which separate resonators pursue their own aims at the respective frequencies, resulting in a simplified and more efficient matching network. Sample heating at the 1H frequency is minimized by a loop-gap resonator which produces a homogeneous magnetic field B 1 with low electric field E. Within the loop-gap resonator, a multi-turn solenoid closely matching the shape of the sample serves as an efficient observe coil. We compare power dissipation in a typical lossy bilayer sample in the new low-E probe and in a previously reported 15N– 1H probe which uses a double-tuned 4-turn solenoid. RF loss in the sample is measured in each probe by observing changes in the 1H 360° pulse lengths. For the same values of 1H B 1 field, sample heating in the new probe was found to be smaller by an order of magnitude. Applications of the low-E design to the PISEMA study of membrane proteins in their native hydrated bilayer environment are demonstrated at 600 and 900 MHz.

Modular Automation Platforms: A Case Study of a Flexible NMR Sample Preparation Robot

A variety of features used by our Department in the design and integration of Automation Platforms are presented here. A challenging project for any automation group is the automation of NMR sample preparation. The dispensing of highly volatile or viscous solutions into the typical 5 mm ID NMR glass tube, and the subsequent capping of the tube, presents unique problems. An angled incremental single-channel dispensing technique prevents bubble formation when a 10-mM protein—based solute is used. A novel gripper finger design, used in conjunction with in-house fabricated Teflon caps, allows reliable capping of NMR tubes. In situ vortexing minimizes vial handling with increased throughput. Magnetic mounting of robot tools (hands) provides precise snap-in positioning with collision-safe breakaway. This simplifies crash recovery during development testing and production use. A wraparound Safety Enclosure with modular safety circuit fulfills ANSI/RIA R15.06–1999 Safety Requirements. Flexible control software permits run interruption for loading and preparation of additional NMR tubes. Prepared samples may be removed during run interruption. A “Fly-By” barcode scanning tool enables positive compound sample ID with improved throughput. Preexisting instrument control software is conveniently interfaced to a Scheduler application through an open-architecture instrument integration framework. This framework allows the development of automation platform—independent Middleware for schedule and assay portability. A new generation of low-power, lightweight, portable and expandable platforms is also presented where a building block tandem approach is used in conjunction with the Rent-a-robot concept for robot recycling.

Investigating macromolecules inside cultured and injected cells by in-cell NMR spectroscopy

The noninvasive character of NMR spectroscopy, combined with the sensitivity of the chemical shift, makes it ideally suited to investigate the conformation, binding events and dynamics of macromolecules inside living cells. These 'in-cell NMR' experiments involve labeling the macromolecule of interest with a nonradioactive but NMR-active isotope (15N or 13C). Cellular samples are prepared either by selectively overexpressing the protein in suitable cells (e.g., bacterial cells grown on isotopically labeled media), or by injecting isotopically labeled proteins directly into either cells or cell extracts. Here we provide detailed protocols for in-cell NMR experiments in the prokaryotic organism Escherichia coli, as well as eukaryotic cells and extracts employing Xenopus laevis oocytes or egg extracts. In-cell NMR samples with proteins overexpressed in E. coli can be produced within 13-14 h. Preparing Xenopus oocyte samples for in-cell NMR experiments takes 6-14 h depending on the oocyte preparation scheme and the injection method used.

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a 13C'-coupled 2D 15N-1H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH2 groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.

On the physical interpretation of density operators at the atomic scale: A thorough analysis of some simple cases

AbstractThe physical meaning of the concepts underlying the density operator formalism are analyzed from different points of view. In particular, the diversity of ways of expressing the density operator of the simplest NMR sample is exploited to show that there are many molecular‐scale physical pictures compatible with each macroscopic state, some of them being more useful than others for specific aims. Those corresponding to diagonal representations of the density matrix conform closely to classical‐like pictures, which allow us to ignore the subtle effects of quantum interferences that are implicit in the concept of coherence. A widespread biconical picture that does not rely on a sound physical basis is shown to be quantitatively valid provided that ad hoc populations are chosen for the involved quantum states. The interpretation of the coefficients of the density operator expansion in terms of observable‐related basis elements is discussed to show that the identification of these with the corresponding physical properties can be misleading in certain cases. © 2006 Wiley Periodicals, Inc. Concepts Magn Reson Part A 28A: 384–409, 2006

Properties of a novel hard-carbon optimized to large size Li ion secondary battery studied by 7Li NMR

The state of lithium in a novel hard-carbon optimized to the anode of large size Li ion secondary battery, which has been recently commercialized, was investigated and compared with other existing hard-carbon samples by 7Li NMR method. The new carbon material showed a peak at 85 ppm with a shoulder signal at 7 ppm at room temperature in static NMR spectrum, and the former shifted to 210 ppm at 180 K. The latter at room temperature was attributed to Li doped in small particles contained in the sample. The new carbon sample showed weaker intensity of cluster-lithium signal than the other hard-carbon samples in NMR, which corresponded to a tendency of less “constant voltage” (CV) capacity in charge–discharge curves of electrochemical evaluation. Smaller CV capacity and initial irreversible capacity, which are the features of the novel hard-carbon, are considered to correspond to a blockade of the diffusion of Li into pore of carbon.

Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Structure and Dynamics of ASC2, a Pyrin Domain-only Protein That Regulates Inflammatory Signaling

Prion Protein Helix1 Promotes Aggregation but Is Not Converted into β-Sheet

Prion diseases are caused by the aggregation of the native alpha-helical prion protein PrP(C) into its pathological beta-sheet-rich isoform PrP(Sc). In current models of PrP(Sc), helix1 is assumed to be preferentially converted into beta-sheet during aggregation of PrP(C). This was supported by the NMR structure of PrP(C) since, in contrast to the isolated helix1, helix2 and helix3 are connected by a small loop and are additionally stabilized by an interhelical disulfide bond. However, helix1 is extremely hydrophilic and has a high helix propensity. This prompted us to investigate the role of helix1 in prion aggregation using humPrP(23-159) including helix1 (144-156) compared with the C-terminal-truncated isoform humPrP(23-144) corresponding to the pathological human stop mutations Q160Stop and Y145Stop, respectively. Most unexpectedly, humPrP(23-159) aggregated significantly faster compared with the truncated fragment humPrP(23-144), clearly demonstrating that helix1 is involved in the aggregation process. However, helix1 is not resistant to digestion with proteinase K in fibrillar humPrP(23-159), suggesting that helix1 is not converted to beta-sheet. This is confirmed by Fourier transformation infrared spectroscopy since there is almost no difference in beta-sheet content of humPrP(23-159) fibrils compared with humPrP(23-144). In conclusion, we provide strong direct evidence that in contrast to earlier assumptions helix1 is not converted into beta-sheet during aggregation of PrP(C) to PrP(Sc).

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP

The First Structure from the SOUL/HBP Family of Heme-binding Proteins, Murine P22HBP