Atomic-level Characterization Research Articles

Magic angle spinning NMR spectroscopy: a versatile technique for structural and dynamic analysis of solid-phase systems.

Magic Angle Spinning (MAS) NMR spectroscopy is a powerful method for analysis of a broad range of systems, including inorganic materials, pharmaceuticals, and biomacromolecules. The recent developments in MAS NMR instrumentation and methodologies opened new vistas to atomic-level characterization of a plethora of chemical environments previously inaccessible to analysis, with unprecedented sensitivity and resolution.

Epitope mapping by solution NMR spectroscopy

Antibodies play an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their epitopes, that is, the region that they recognize on their target molecule, is useful for purposes ranging from molecular biology research to vaccine design and intellectual property protection. Solution NMR spectroscopy is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to epitope discovery. Here, we illustrate how NMR epitope mapping can be used to rapidly and accurately determine protein antigen epitopes. The basic concept is that differences in the NMR signal of an antigen free or bound by an antibody will identify epitope residues. NMR epitope mapping provides more detailed information than mutagenesis or peptide mapping and can be much more rapid than X-ray crystallography. Advantages and drawbacks of this technique are discussed together with practical considerations.

Atomic-Level Characterization of the Inhibition Mechanism of the Calcium Pump by Phospholamban

We have performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca2+-ATPase, SERCA) in complex with phospholamban (PLB). Regulation of SERCA activity by PLB is central in modulating cardiac contractility. X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca2+ affinity E2 state that is incompatible with metal ion binding, thereby blocking the conversion toward a high-Ca2+ affinity E1 state. Estimation of pKa values of the acidic residues in the Ca2+ sites indicates that under normal intracellular pH (7.1-7.2), PLB-bound SERCA populates an E1 state deprotonated at residues E309 and D800 yet protonated at residue E771. We performed microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K+ and 3 mM Mg2+. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol, and that two K+ ions bind transiently (≤5 ns), and non-specifically (9 different positions) to the two transport sites; the total occupancy time of the two K+ ions in the transport sites is 80%. These findings indicate that PLB does not inhibit the E2-to-E1 interconversion but instead populates a novel E1 intermediate, E1•H+771, that depresses SERCA activation by Ca2+. We propose that the efficient regulation of SERCA activity by PLB results from structural transitions that occur primarily in the E1 state of the pump. This work was supported by grants to L.M.E-F. from the American Heart Association (12SDG12060656) and to D.D.T from NIH (GM27906), and by the University of Minnesota Supercomputing Institute.

Conjugated precipitation of twin-related α and Ti2Cu phases in a Ti–25V–3Cu alloy

The formation of conjugated precipitates in a ternary Ti–25V–3Cu (wt.%) (Ti–24.4V–2.3Cu (at.%)) alloy subjected to isothermal aging at 500°C was investigated in this study. X-ray diffraction confirms that the equilibrium precipitates in the alloy are hexagonal α phase and body-centered-tetragonal (bct) Ti2Cu intermetallic compound. These precipitates are composed of two geometrically symmetrical components, which are verified by transmission electron microscopy (TEM) to be twin-related variants of α and Ti2Cu phases. Atomic-level characterizations incorporating high-resolution TEM and atom probe tomography were used to investigate the early-stage nucleation process of the precipitates. The results reveal that precipitation occurs heterogeneously on plate-like (or disc-like) metastable Cu-enriched GP zones that preferentially inhabit {110}β planes. Crystallographic orientation analyses suggest that the Ti2Cu phase is related to the matrix via a definite orientation relationship of (01¯3)Ti2Cu‖(011)β and [100]Ti2Cu‖[100]β, while the growth of the α phase is governed by the Potter’s OR such that (11¯01)α‖(011)β and [112¯0]α‖[11¯1]β. (01¯3)Ti2Cu and (11¯01)α are the twin planes of the respective phases. In spite of highly distinct crystal structures, the conjugated α and Ti2Cu phases attain a nearly perfect lattice correspondence along certain crystallographic planes, which results in a low interfacial energy configuration and favours the co-development of these phases.

Simulating chemical reactions in ionic liquids using QM/MM methodology.

The use of ionic liquids as a reaction medium for chemical reactions has dramatically increased in recent years due in large part to the numerous reported advances in catalysis and organic synthesis. In some extreme cases, ionic liquids have been shown to induce mechanistic changes relative to conventional solvents. Despite the large interest in the solvents, a clear understanding of the molecular factors behind their chemical impact is largely unknown. This feature article reviews our efforts developing and applying mixed quantum and molecular mechanical (QM/MM) methodology to elucidate the microscopic details of how these solvents operate to enhance rates and alter mechanisms for industrially and academically important reactions, e.g., Diels-Alder, Kemp eliminations, nucleophilic aromatic substitutions, and β-eliminations. Explicit solvent representation provided the medium dependence of the activation barriers and atomic-level characterization of the solute-solvent interactions responsible for the experimentally observed "ionic liquid effects". Technical advances are also discussed, including a linear-scaling pairwise electrostatic interaction alternative to Ewald sums, an efficient polynomial fitting method for modeling proton transfers, and the development of a custom ionic liquid OPLS-AA force field.

NMR crystallography, diffraction and modeling of small organic molecules

Solid-state NMR (SSNMR) is a powerful atomic-level characterization technique able to study the local chemical environment of a nucleus in crystalline/amorphous solids. Toward a better understanding of how small molecules self-assemble in the solid-state and reorganizes to produce its hydrate/anhydrous forms, an experimental SSNMR, X-ray diffraction (XRD), and computational study of the supramolecular assemblies of selected small pharmaceuticals is presented. The effect of crystal packing on the 1H and 13C chemical shifts including nonconventional hydrogen bonds, pi···pi and CH···pi contacts, is studied through computer simulations. It will be shown that NMR chemical shifts are sensitive detectors of hydration/dehydration states in highly insoluble antibiotics.[1] Recently, SSNMR became an important gadget in the process of crystal structure solution in powders. This is a non-trivial task and using powder XRD methods alone may often lead to the wrong structure solution. In this talk, a new hybrid approach for structure determination of crystalline solids, will be presented, based on the combination of SSNMR, XRD and an ensemble of computational-assisted structure solution tools including a genetic algorithm based on evolution-inspired operators repeatedly applied to populations of possible crystal structure solutions that evolve to eventually produce the best new offspring candidates. Such methodologies are successfully applied to challenging cases involving multiple component crystals composed by flexible molecules such as a trihydrate β-lactamic antibiotic [2] and an azole-based co-crystal featuring an hydrogen bond network of α-helixes involving NH···N/CH···π intermolecular interactions. ACKNOLEDGEMENTS: Supported by Fundação para a Ciência e a Tecnologia (FCT), Portuguese National NMR Network (RNRMN), CICECO (PEst-C/CTM/LA0011/2013), FEDER, COMPETE, and University of Aveiro. FCT is greatly acknowledge for the consolidation grant IF/01401/2013.

In situ ETEM synthesis of NiGa alloy nanoparticles from nitrate salt solution.

Metallic alloy nanoparticles (NPs) are synthesized in situ in an environmental transmission electron microscope. Atomic level characterization of the formed alloy NPs is carried out at synthesis conditions by use of high-resolution transmission electron microscopy, electron diffraction and electron energy-loss spectroscopy.

On the stability of metadislocations with 16 associated phason planes

High-resolution scanning transmission electron microscopy was used to investigate the core structure of a metadislocation in a plastically deformed sample of the complex metallic alloy ε6-Al–Pd–Mn. The atomic-level characterization of a metadislocation with 16 associated phason planes showed dissociation into metadislocations with 6 and 10 phason planes. A structural model of the dissociated core region was developed. The Burgers vector of the dislocation b→16 = −0.07 nm [001] splits up into b→6 = −0.183 nm [001] and b→10 = 0.113 nm [001] increasing the strain at the metadislocation cores. Although unfavourable with respect to an increasing elastic strain energy the dissociation is realized due to a decrease in fault-plane energy. The latter arises from a size reduction of the ξ-phase area that is required to accommodate the metadislocation in the structure. A careful inspection of several exemplary metadislocations with 16 phason planes using electron microscopy indicates their dissociated structure which casts doubt on the stability of elementary metadislocations with 16 phason planes. The study includes a detailed analysis of the structure of a metadislocation core with 6 associated phason planes.

Atomic-Level Characterization of Interfaces in LiCoO2

The powerful synergy between atomic-resolution electron microscopy and atomistic simulation techniques encompassing both first-principles and classical force-field calculations is illustrated through a study of grain boundaries, domain boundaries and surfaces of the lithium-ion secondary battery cathode material LiCoO2. Epitaxial thin films of LiCoO2 deposited on sapphire (0001) substrates are used as model samples, and results from simulation and experiment combined to characterize the structures and properties of representative interfaces.

Dynamic Nuclear Polarization Enhanced NMR Spectroscopy for Pharmaceutical Formulations

Dynamic nuclear polarization (DNP) enhanced solid-state NMR spectroscopy at 9.4 T is demonstrated for the detailed atomic-level characterization of commercial pharmaceutical formulations. To enable DNP experiments without major modifications of the formulations, the gently ground tablets are impregnated with solutions of biradical polarizing agents. The organic liquid used for impregnation (here 1,1,2,2-tetrachloroethane) is chosen so that the active pharmaceutical ingredient (API) is minimally perturbed. DNP enhancements (ε) of between 40 and 90 at 105 K were obtained for the microparticulate API within four different commercial formulations of the over-the-counter antihistamine drug cetirizine dihydrochloride. The different formulations contain between 4.8 and 8.7 wt % API. DNP enables the rapid acquisition with natural isotopic abundances of one- and two-dimensional (13)C and (15)N solid-state NMR spectra of the formulations while preserving the microstructure of the API particles. Here this allowed immediate identification of the amorphous form of the API in the tablet. API-excipient interactions were observed in high-sensitivity (1)H-(15)N correlation spectra, revealing direct contacts between povidone and the API. The API domain sizes within the formulations were determined by measuring the variation of ε as a function of the polarization time and numerically modeling nuclear spin diffusion. Here we measure an API particle radius of 0.3 μm with a single particle model, while modeling with a Weibull distribution of particle sizes suggests most particles possess radii of around 0.07 μm.

A new approach to atomic level characterization of grain boundaries by atom probe tomography

ABSTRACTA novel atom probe tomography (APT) method has been developed that enables a full description of the orientation relationship between individual grains to be determined together with estimates of the extents of solute segregation for all elements over the surface of the grain boundary with 1 nm by 1 nm spatial resolution. This approach also enables variations in the solute excess for the elements with the habit plane and curvature of the grain boundary to be evaluated. The method has been applied to a mechanically-alloyed nanostructured ferritic alloy (NFA) after high dose heavy ion irradiation. The innovative high-resolution two-dimensional mapping of the solute segregation across the surface of grain boundaries in the NFA clearly demonstrates that the distributions of chromium and tungsten are not uniform across the grain boundaries, and the distributions correlate with changes in its local curvature and the position of the grain boundary precipitates. These features pin the grain boundary against grain growth and provide the stability for excellent creep properties.

Accelerated Monte-Carlo Simulations for All-Atom Protein Folding

Recent advances in the computational description of proteins facilitate the atomic-level characterization of fast folding proteins using molecular-dynamics simulations[1]. However, due to the femtosecond timestep of an MD simulation, 10 ˆ 10 computationally expensive energy evaluations are required to reach timescales of the order of single folding events even for ultrafast-folding proteins. Stochastic simulation techniques can overcome this limiting factor by generating thermodynamic conformational ensembles without incorporating atomic vibrations, but most of these methods are inherently sequential. Here we investigate a parallel extension to the conventional Metropolis-Hastings algorithm, the Multiple-Try-Metropolis, enabling parallel simulation of reversible Markov-chains[2,3]. We successfully characterize the thermodynamic landscape of the Villin-headpiece-subdomain using a generalized MTM approach adapted to protein systems. We parameterized the polypeptide using the AMBER99SB-STAR-ILDN-forcefield with an implicit solvent model. In our simulations we could observe approximately two folding transitions per day on standard hardware, with a total of 109 transition events during 3∗10 ˆ 9 energy evaluations. The MTM approach thus facilitates the efficient thermodynamic characterization of proteins on common computer architectures. The comparison with experimental resolved folding times of 8µs translates to a time equivalent of 16ps per MTM step.[1] Shaw, Science-2010.[2] Liu, JASA-2000.[3] Pandolfi, JMLR-2010.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Atomic-Level Characterization of Interfaces in LiCoO2

not Available.

Interaction of the Eukaryotic Initiation Factor 4E with 4E-BP2 at a Dynamic Bipartite Interface

Cap-dependent translation initiation is regulated by the interaction of eukaryotic initiation factor 4E (eIF4E) with eIF4E binding proteins (4E-BPs). Whereas the binding of 4E-BP peptides containing the eIF4E-binding ⁵⁴YXXXXLΦ⁶⁰ motif has been studied, atomic-level characterization of the interaction of eIF4E with full-length 4E-BPs has been lacking. Here, we use isothermal titration calorimetry and nuclear magnetic resonance spectroscopy to characterize the dynamic, structural and binding properties of 4E-BP2. Although disordered, 4E-BP2 contains significant fluctuating secondary structure and binds eIF4E at an extensive bipartite interface including the canonical ⁵⁴YXXXXLΦ⁶⁰ and ⁷⁸IPGVT⁸² sites. Each of the two binding elements individually has submicromolar affinity and exchange on and off of the eIF4E surface within the context of the overall nanomolar complex. This dynamic interaction facilitates exposure of regulatory phosphorylation sites within the complex. The 4E-BP2 interface on eIF4E overlaps yet is more extensive than the eIF4G:eIF4E interface, suggesting that these key interactions may be differentially targeted for therapeutics.

Atomic level characterization and sulfur resistance of unsupported W2C during dibenzothiophene hydrodesulfurization. Classical kinetic simulation of the reaction

The stability of unsupported W2C tungsten carbide and its sulfur resistance during hydrodesulfurization (HDS) of dibenzothiophene (DBT) are a specificity of W2C. This material was characterized by X-ray diffraction, specific surface area measurements, HRTEM associated with EDS, lattice images, direct and reverse Fast Fourier Transforms, direct lattice fringes profiles, dynamic CO chemisorptions and elemental analysis of S, C and W. The absence of surface crystallographic layer of tungsten sulfide and the presence of a surface monolayer of “adsorbed sulfur species” (post-sulfur passivation by H2S by the end of HDS runs), after 5 days of HDS runs on unsupported W2C, are established. The repeatability of both W2C synthesis and catalytic activity with chemical stability are shown. The HDS of DBT was investigated using a fixed-bed reactor. The reaction was carried out at 613K, under a 6MPa total pressure of H2. The global reaction follows two parallel routes. Under the experimental conditions, DBT has a single final product along each route, leading either to biphenyl through the direct desulfurization (DDS) pathway, or to cyclohexylbenzene through the hydrogenation (HYD) pathway. Accordingly, the HDS of DBT over W2C presents a true zero order reaction to DBT along the DDS route, and a true first order reaction along the HYD route. The resulting global apparent first order rate with respect to DBT is linked to the selectivity towards these two routes. The kinetically true zero order rate along the DDS route corresponds to the saturation by DBT of all its active sites and, consequently, means that the HYD route is occurring on a second kind of sites. The presence of two kinds of sites is thus kinetically demonstrated and discussed in term of Coordinatively Unsaturated Sites (CUS).

Computationally Efficient Methodology for Atomic-Level Characterization of Dendrimer–Drug Complexes: A Comparison of Amine- and Acetyl-Terminated PAMAM

PAMAM dendrimers have been widely studied as a novel means for controlled drug delivery; however, computational study of dendrimer-drug complexation is made difficult by the conformational flexibility of dendrimers and the nonspecific nature of the dendrimer-drug interactions. Conventional protocols for studying drug binding have been designed primarily for protein substrates, and, therefore, there is a need to establish new protocols to deal with the unique aspects of dendrimers. In this work, we generate cavities in generation-5 polyamidoamine (PAMAM) dendrimers at selected distances from the center of mass of the dendrimer for the insertion of the model drug: dexamethasone 21-phosphate or Dp21. The complexes are then allowed to equilibrate with distance between centers of mass of the drug and dendrimers confined to selected ranges; the free energy of complexation is estimated by the MM-GBSA (MM, molecular mechanics; GB, generalized Born; SA, surface area) method. For both amine- and modified acetyl-terminated PAMAM at both low and neutral pH, the most favorable free energy of complexation is associated with Dp21 at distance of 15-20 Å from the center of mass of the dendrimer and that smaller or larger distances yield considerably weaker affinity. In agreement with experimental results, we find acetyl-terminated PAMAM at neutral pH to form the least stable complex with Dp21. The greatest affinity is seen in the case of acetyl-terminated PAMAM at low pH, which appears to be due a complex balance of different contributions, which cannot be attributed to electrostatics, van der Waals interactions, hydrogen bonds, or charge-charge interactions alone.

Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy

Many of the functions and applications of advanced materials result from their interfacial structures and properties. However, the difficulty in characterizing the surface structure of these materials at an atomic level can often slow their further development. Solid-state NMR can probe surface structure and complement established surface science techniques, but its low sensitivity often limits its application. Many materials have low surface areas and/or low concentrations of active/surface sites. Dynamic nuclear polarization (DNP) is one intriguing method to enhance the sensitivity of solid-state NMR experiments by several orders of magnitude. In a DNP experiment, the large polarization of unpaired electrons is transferred to surrounding nuclei, which provides a maximum theoretical DNP enhancement of ∼658 for (1)H NMR. In this Account, we discuss the application of DNP to enhance surface NMR signals, an approach known as DNP surface enhanced NMR spectroscopy (DNP SENS). Enabling DNP for these systems requires bringing an exogeneous radical solution into contact with surfaces without diluting the sample. We proposed the incipient wetness impregnation technique (IWI), a well-known method in materials science, to impregnate porous and particulate materials with just enough radical containing solution to fill the porous volume. IWI offers several advantages: it is extremely simple, provides a uniform wetting of the surface, and does not increase the sample volume or substantially reduce the concentration of the sample. This Account describes the basic principles behind DNP SENS through results obtained for mesoporous and nanoparticulate samples impregnated with radical solutions. We also discuss the quantification of the overall sensitivity enhancements obtained with DNP SENS and compare that with ordinary room temperature NMR spectroscopy. We then review the development of radicals and solvents that give the best possible enhancements today. With the best polarizing mixtures, DNP SENS enhances sensitivity by a factor of up to 100, which decreases acquisition time by five orders of magnitude. Such enhancement enables the detailed and expedient atomic level characterization of the surfaces of complex materials at natural isotopic abundance and opens new avenues for NMR. To illustrate these improvements, we describe the successful application of DNP SENS to characterize hybrid materials, organometallic surface species, and metal-organic frameworks.

Intrinsic Protein Disorder in the Regulation of Large Molecular Machines

We here report the molecular overlap of the linkage of three essential protein complexes that coordinate the formation of the mitotic spindle. These proteins are dynein, a large motor complex that moves machinery inside cells, and two of its regulators: a protein complex called dynactin, that is a dynein activator, and a protein called NudE whose depletion in mice produces a small brain and mental retardation. What is intriguing about the dynein/dynactin/NudE interplay is that dynactin and NudE bind to a common segment of dynein that is intrinsically disordered but with distinct binding modes. Elucidating details of these distinct modes explains how one regulator is selected over the other even when both are present in the same cellular compartment. Using NMR spectroscopy and isothermal titration calorimetry we show that intrinsic disorder in a specific segment of dynein intermediate chain promotes local modifications like phosphorylation and splicing, promotes rapid equilibration of ensemble components in solution samples of complexes with moderate to weak binding affinities, and creates a bi-segmental binding site such that residue-level modification in and near one segment have minimal effect on the structure of the second segment thereby aiding in segregating of functions between two consecutive segments. These results underscore the role of disorder in the versatility of dynein binding to different regulators, and have far reaching impact not only on our understanding of processes essential for formation and orientation of the spindle, but also offer a novel role for protein disorder in controlling cellular processes, and highlight the advantages of NMR spectroscopy in elucidating atomic level characterization of extremely complex dynamic cellular assemblies.

Native disorder mediates binding of dynein to NudE and dynactin

In the present paper, I report the molecular overlap of the linkage of three essential protein complexes that co-ordinate the formation of the mitotic spindle. These proteins are dynein, a large motor complex that moves machinery inside cells, and two of its regulators: a protein complex called dynactin, a dynein activator, and a protein called NudE whose depletion in mice produces a small brain and mental retardation. What is intriguing about the dynein-dynactin-NudE interplay is that dynactin and NudE bind to a common segment of dynein that is intrinsically disordered. Elucidating differences in their binding modes may explain how one regulator can be selected over the other even when both are present in the same cellular compartment. These results not only have a far-reaching impact on our understanding of processes essential for the formation and orientation of the spindle, but also offer a novel role for protein disorder in controlling cellular processes, and highlight the advantages of NMR spectroscopy in elucidating atomic-level characterization of extremely complex dynamic cellular assemblies.

5th Congress of the International Union of Microbeam Analysis Societies in conjunction with the 8th International Symposium on Atomic Level Characterization for New Materials and Devices' 11

Surface and Interface AnalysisVolume 44, Issue 11-12 p. 1385-1385 Preface 5th Congress of the International Union of Microbeam Analysis Societies in conjunction with the 8th International Symposium on Atomic Level Characterization for New Materials and Devices' 11 Gyeung-Ho Kim, Corresponding Author Gyeung-Ho Kim Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, KoreaAdvanced Analysis Center, Korea Institute of Science and Technology, Seoul KoreaSearch for more papers by this authorJon-Do Yun, Jon-Do Yun Department of Nano Science and Engineering, Kyungnam University, Masan, KoreaSearch for more papers by this authorSe-Ahn Song, Se-Ahn Song Computational and Analytical Science Center, Samsung Advanced Institute of Technology, Suwon, KoreaSearch for more papers by this author Gyeung-Ho Kim, Corresponding Author Gyeung-Ho Kim Advanced Analysis Center, Korea Institute of Science and Technology, Seoul, KoreaAdvanced Analysis Center, Korea Institute of Science and Technology, Seoul KoreaSearch for more papers by this authorJon-Do Yun, Jon-Do Yun Department of Nano Science and Engineering, Kyungnam University, Masan, KoreaSearch for more papers by this authorSe-Ahn Song, Se-Ahn Song Computational and Analytical Science Center, Samsung Advanced Institute of Technology, Suwon, KoreaSearch for more papers by this author First published: 07 September 2012 https://doi.org/10.1002/sia.5166Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume44, Issue11-12Special Issue: 5th Congress of the International Union of Microbeam Analysis Societies in conjunction with the 8th International Symposium on Atomic Level Characterization for New Materials and Devices' 11November-December 2012Pages 1385-1385 RelatedInformation