Application In Structural Studies Research Articles

Methods and application of coherent X-ray diffraction imaging of noncrystalline particles.

Microscopic imaging techniques have been developed to visualize events occurring in biological cells. Coherent X-ray diffraction imaging is one of the techniques applicable to structural analyses of cells and organelles, which have never been crystallized. In the experiment, a single noncrystalline particle is illuminated by an X-ray beam with almost complete spatial coherence. The structure of the particle projected along the direction of the beam is, in principle, retrieved from a finely recorded diffraction pattern alone by using iterative phase-retrieval algorithms. Here, we describe fundamental theory and experimental methods of coherent X-ray diffraction imaging and the recent application in structural studies of noncrystalline specimens by using X-rays available at Super Photon Ring of 8-Gev and SPring-8 Angstrom Compact Free Electron Laser in Japan.

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrPSc) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrPSc under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40]3– ion decomposes to give a lacunary [PW11O39]7– (PW11) complex and a single orthotungstate anion [WO4]2– (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrPSc from brain homogenates of scrapie prion-infected mice. The amount of PrPSc isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrPSc precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrPSc were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrPSc in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrPSc aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrPSc. We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrPSc by PTA from tissue and might be optimized further for the detection of prions.

Gravitational Gradients at Satellite Altitudes in Global Geophysical Studies

Due to the ESA’s satellite mission GOCE launched in March 2009, gravitational gradients sampled along the orbital trajectory approximately 250 km above the Earth’s surface have become available. Since 2010, gravitational gradients have routinely been applied in geodesy for the derivation of global Earth’s gravitational models provided in terms of fully normalized coefficients in a spherical harmonic series representation of the Earth’s gravitational potential. However, in geophysics, gravitational gradients observed by spaceborne instruments have still been applied relatively seldom. This contribution describes their possible geophysical applications in structural studies where gravitational gradients observed at satellite altitudes are compared with those derived by a spectral forward modeling technique using available global models of selected Earth’s mass components as input data. In particular, GOCE gravitational gradients are interpreted in terms of a superposition principle of gravitation as combined gravitational effects generated by a homogeneous reference ellipsoid of revolution, mean topographic and ice mass density distributions, depth-dependent mass density contrasts within bathymetry and lateral mass density anomalies with sediments and crustal layers. Respective gravitational effects are one by one removed from gravitational gradients observed at approximately 250 km elevation above ground. Removing respective gravitational gradients from observed gravitational gradients gradually reveals problematic geographic areas with model deficiencies. For the full interpretation of observed gravitational gradients, deficiencies of CRUST2.0 must be corrected and effects of deeper laying mass anomalies not included in the study considered. These findings are confirmed by parameters describing spectral properties of the gravitational gradients. The methodology can be applied for validating Earth’s gravitational models and for constraining crustal models in the development phase.

Imino hydrogen positions in nucleic acids from density functional theory validated by NMR residual dipolar couplings.

Hydrogen atom positions of nucleotide bases in RNA structures solved by X-ray crystallography are commonly derived from heavy-atom coordinates by assuming idealized geometries. In particular, N1–H1 vectors in G and N3–H3 vectors in U are commonly positioned to coincide with the bisectors of their respective heavy-atom angles. We demonstrate that quantum-mechanical optimization of the hydrogen positions relative to their heavy-atom frames considerably improves the fit of experimental residual dipolar couplings to structural coordinates. The calculations indicate that deviations of the imino N–H vectors in RNA U and G bases result from H-bonding within the base pair and are dominated by the attractive interaction between the H atom and the electron density surrounding the H-bond-acceptor atom. DFT optimization of H atom positions is impractical in structural biology studies. We therefore have developed an empirical relation that predicts imino N–H vector orientations from the heavy-atom coordinates of the base pair. This relation agrees very closely with the DFT results, permitting its routine application in structural studies.

Evaluation of the Pichia pastoris expression system for the production of GPCRs for structural analysis

BackgroundVarious protein expression systems, such as Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), Pichia pastoris (P. pastoris), insect cells and mammalian cell lines, have been developed for the synthesis of G protein-coupled receptors (GPCRs) for structural studies. Recently, the crystal structures of four recombinant human GPCRs, namely β2 adrenergic receptor, adenosine A2a receptor, CXCR4 and dopamine D3 receptor, were successfully determined using an insect cell expression system. GPCRs expressed in insect cells are believed to undergo mammalian-like posttranscriptional modifications and have similar functional properties than in mammals. Crystal structures of GPCRs have not yet been solved using yeast expression systems. In the present study, P. pastoris and insect cell expression systems for the human muscarinic acetylcholine receptor M2 subtype (CHRM2) were developed and the quantity and quality of CHRM2 synthesized by both expression systems were compared for the application in structural studies.ResultsThe ideal conditions for the expression of CHRM2 in P. pastoris were 60 hr at 20°C in a buffer of pH 7.0. The specific activity of the expressed CHRM2 was 28.9 pmol/mg of membrane protein as determined by binding assays using [3H]-quinuclidinyl benzilate (QNB). Although the specific activity of the protein produced by P. pastoris was lower than that of Sf9 insect cells, CHRM2 yield in P. pastoris was 2-fold higher than in Sf9 insect cells because P. pastoris was cultured at high cell density. The dissociation constant (Kd) for QNB in P. pastoris was 101.14 ± 15.07 pM, which was similar to that in Sf9 insect cells (86.23 ± 8.57 pM). There were no differences in the binding affinity of CHRM2 for QNB between P. pastoris and Sf9 insect cells.ConclusionCompared to insect cells, P. pastoris is easier to handle, can be grown at lower cost, and can be expressed quicker at a large scale. Yeast, P. pastoris, and insect cells are all effective expression systems for GPCRs. The results of the present study strongly suggested that protein expression in P. pastoris can be applied to the structural and biochemical studies of GPCRs.

Fluoride-mediated capture of a noncovalent bound state of a reversible covalent enzyme inhibitor: X-ray crystallographic analysis of an exceptionally potent α-ketoheterocycle inhibitor of fatty acid amide hydrolase.

Two cocrystal X-ray structures of the exceptionally potent α-ketoheterocycle inhibitor 1 (K(i) = 290 pM) bound to a humanized variant of rat fatty acid amide hydrolase (FAAH) are disclosed, representing noncovalently and covalently bound states of the same inhibitor with the enzyme. Key to securing the structure of the noncovalently bound state of the inhibitor was the inclusion of fluoride ion in the crystallization conditions that is proposed to bind the oxyanion hole precluding inhibitor covalent adduct formation with stabilization of the tetrahedral hemiketal. This permitted the opportunity to detect important noncovalent interactions stabilizing the binding of the inhibitor within the FAAH active site independent of the covalent reaction. Remarkably, noncovalently bound 1 in the presence of fluoride appears to capture the active site in the same "in action" state with the three catalytic residues Ser241-Ser217-Lys142 occupying essentially identical positions observed in the covalently bound structure of 1, suggesting that this technique of introducing fluoride may have important applications in structural studies beyond inhibiting substrate or inhibitor oxyanion hole binding. Key insights to emerge from the studies include the observations that noncovalently bound 1 binds in its ketone (not gem diol) form, that the terminal phenyl group in the acyl side chain of the inhibitor serves as the key anchoring interaction overriding the intricate polar interactions in the cytosolic port, and that the role of the central activating heterocycle is dominated by its intrinsic electron-withdrawing properties. These two structures are also briefly compared with five X-ray structures of α-ketoheterocycle-based inhibitors bound to FAAH recently disclosed.

Raman tensors and their application in structural studies of biological systems.

The Raman scattering of a molecule is generated by interactions of its electrons with incident light. The electric vector of the Raman scattered light is related to the electric vector of the incident light through a characteristic Raman tensor. A unique Raman tensor exists for each Raman-active molecular vibrational mode. In the case of biologically important macromolecules Raman tensors have been determined for a few hundred vibrational Raman bands. These include proteins and their amino acid constituents, as well as nucleic acids (DNA and RNA) and their nucleotide constituents. In this review Raman tensors for 39 representative vibrational Raman bands of biological molecules are considered. We present details of the Raman tensor determinations and discuss their application in structural studies of filamentous bacteriophages (fd, Pf1, Pf3 and PH75), fowl feather rachis and eyespots of the protists, Chlamydomonas and Euglena.

Construction of fluorescence resonance energy transfer vectors and their application in study of structure and function of signal transducers and activators of transcription 1

Protein-protein interactions have been studied extensively by green fluorescent protein-based fluorescence resonance energy transfer (FRET). The fluorescent proteins (FP) can be fused either to the N- or C-terminus of a host protein, but it is difficult to predict which order will perturb the host protein the least and provide the largest FRET. Therefore, a researcher needs to fuse host proteins with FP at both the N- and C-termini and test every possible combination (N-N, N-C, or C-C) to promote the energy transfer efficiency. Consequently, researchers required to do many subclonings. Herein, we designed FRET vectors to make them more efficient. The expression vectors of pCFP-YFP and pYFP-CFP were constructed with both cyan fluorescent protein (CFP)-yellow fluorescent protein (YFP) and YFP-CFP coding sequences flanked by two restriction enzyme sites, and with multiple cloning regions in the middle of both coding sequences. To select an optimal combination for FRET detection, we created plasmids encoding various fusion proteins of FP and signal transducers and activators of transcription 1 (STAT1) . We found that the nuclear:cytoplasmic fluorescence intensity ratios of STAT1-FP were significantly higher than those of FP-STAT1 at steady state, and fluorescence redistribution was only observed for STAT1-FP upon interferon gamma (IFNgamma) stimulation. In addition, positive FRET signals were only detected in the C-C interactions of STAT1 homodimer. Taken together, these data indicate that fusing STAT1 at the N-terminus with FP impairs the interactions of unphosphorylated STAT1 homodimers and possibly diminishes its binding with DNA. In contrast, STAT1-FP was functional with respect to its activation. Moreover, the FRET vectors are able to facilitate FRET studies.

Rotational resonance in uniformly 13C-labeled solids: effects on high-resolution magic-angle spinning NMR spectra and applications in structural studies of biomolecular systems

We describe investigations of the effects of rotational resonance ( R 2) on solid state 13C NMR spectra of uniformly 13C-labeled samples obtained under magic-angle spinning (MAS), and of the utility of R 2 measurements as structural probes of peptides and proteins with multiple uniformly labeled residues. We report results for uniformly 13C-labeled l-alanine and l-valine in polycrystalline form, and for amyloid fibrils formed by the 15-residue peptide Aβ 11–25 with uniform labeling of a four-residue segment. The MAS NMR spectra reveal a novel J-decoupling effect at R 2 conditions that may be useful in spectral assignments for systems with sharp 13C MAS NMR lines. Pronounced dependences of the apparent isotropic 13C NMR chemical shifts on MAS frequency near R 2 conditions are also observed. We demonstrate the feasibility of quantitative 13C– 13C distance determinations in l-valine, and qualitative determinations of inter-residue 13C– 13C contacts in Aβ 11–25 fibrils. Finally, we demonstrate a “relayed” R 2 technique that may be useful in structural measurements on systems with poorly resolved 13C MAS NMR lines.

The development of a high resolution mass spectrometer: a reminiscence

This article is a review of the events leading to the development of the double-focusing tandem mass spectrometer system, which is sometimes called the Johnson-Nier geometry. An essential feature of the geometry is that it eliminates second-order angular aberration, enhancing the sensitivity, without a loss in resolution. Flexibility in the choice of ion source and collector designs is assured because both are outside of regions of electric and magnetic fields. The vacuum housing permits high-temperature baking, ensuring ultrahigh vacuum conditions. Although introduced initially for the purpose of determining precise atomic masses, the design has found its greatest application in studies of structure of heavy molecules, making use of the high resolution to identify fragments. In many cases the composition of a molecule, or fragment, can be deduced from its exact mass by utilizing the known atomic masses of likely constituent atoms.

Neutron scatter and diffraction techniques applied to nucleosome and chromatin structure

Neutron scatter and diffraction techniques have made substantial contributions to our understanding of the structure of the nucleosome, the structure of the 10-nm filament, the "10-nm----30-nm" filament transition, and the structure of the "34-nm" supercoil or solenoid of nucleosomes. Neutron techniques are unique in their properties, which allows for the separation of the spatial arrangements of histones and DNA in nucleosomes and chromatin. They have equally powerful applications in structural studies of any complex two-component biological system. A major success for the application of neutron techniques was the first clear proof that DNA was located on the outside of the histone octamer in the core particle. A full analysis of the neutron-scatter data gave the parameters of Table 3 and the low-resolution structure of the core particle in solution shown in Fig. 6. Initial low-resolution X-ray diffraction studies of core particle crystals gave a model with a lower DNA pitch of 2.7 nm. Higher-resolution X-ray diffraction studies now give a structure with a DNA pitch of 3.0 nm and a hole of 0.8 nm along the axis of the DNA supercoil. The neutron-scatter solution structure and the X-ray crystal structure of the core particle are thus in full agreement within the resolution of the neutron-scatter techniques. The model for the chromatosome is largely based on the structural parameters of the DNA supercoil in the core particle, nuclease digestion results showing protection of a 168-bp DNA length by histone H1 and H1 peptide, and the conformational properties of H1. The path of the DNA outside the chromatosome is not known, and this information is crucial for our understanding of higher chromatin structure. The interactions of the flexible basic and N- and C-terminal regions of H1 within chromatin and how these interactions are modulated by H1 phosphorylation are not known. The N- and C-terminal regions of H1 represent a new type of protein behavior, i.e., extensive protein domains that are designed not to fold up into secondary and tertiary protein structures. This behavior is increasingly observed in DNA and chromatin binding proteins, and in the case of the high-mobility group proteins HMG 14 and 17, the entire polypeptide chain is a flexible random coil over a wide range of solution, ionic, and pH conditions. It follows that the native conformations are probably imposed on these flexible domains and molecules by their binding sites in chromatin.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiphoton mass spectrometry

ADVERTISEMENT RETURN TO ISSUEPREVArticleMultiphoton mass spectrometryEdward W. Schlag and Hans J. NeusserCite this: Acc. Chem. Res. 1983, 16, 9, 355–360Publication Date (Print):September 1, 1983Publication History Published online1 May 2002Published inissue 1 September 1983https://doi.org/10.1021/ar00093a007RIGHTS & PERMISSIONSArticle Views134Altmetric-Citations79LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (734 KB) Get e-Alerts Get e-Alerts

ChemInform Abstract: ADDITIVITY RELATION IN THE AMPLITUDES OF EXCITON‐SPLIT CIRCULAR DICHROISM CURVES ARISING FROM INTERACTIONS BETWEEN DIFFERENT CHROMOPHORES AND ITS APPLICATION IN STRUCTURAL STUDIES

Chemischer InformationsdienstVolume 14, Issue 16 Physical Organic Chemistry ChemInform Abstract: ADDITIVITY RELATION IN THE AMPLITUDES OF EXCITON-SPLIT CIRCULAR DICHROISM CURVES ARISING FROM INTERACTIONS BETWEEN DIFFERENT CHROMOPHORES AND ITS APPLICATION IN STRUCTURAL STUDIES R. J. STONARD, R. J. STONARDSearch for more papers by this authorD. A. TRAINOR, D. A. TRAINORSearch for more papers by this authorM. NAKATANI, M. NAKATANISearch for more papers by this authorK. NAKANISHI, K. NAKANISHISearch for more papers by this author R. J. STONARD, R. J. STONARDSearch for more papers by this authorD. A. TRAINOR, D. A. TRAINORSearch for more papers by this authorM. NAKATANI, M. NAKATANISearch for more papers by this authorK. NAKANISHI, K. NAKANISHISearch for more papers by this author First published: April 19, 1983 https://doi.org/10.1002/chin.198316051AboutPDF 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 onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume14, Issue16April 19, 1983 RelatedInformation

Additivity relation in the amplitudes of exciton-split circular dichroism curves arising from interactions between different chromophores and its application in structural studies

Additivity relation in the amplitudes of exciton-split circular dichroism curves arising from interactions between different chromophores and its application in structural studies

Characterization of a new photoaffinity derivative of ouabain: labeling of the large polypeptide and of a proteolipid component of the (sodium-potassium ion)-dependent ATPase

We have synthesized 2-nitro-5-azidobenzoyl (NAB) derivatives of ouabain as photoaffinity labels of the cardiac glyocoside binding site of Na, K-ATPase. [3HzNAB-ouabain was found to bind to the same number of sites on Na, K-ATPase (purified from pig kidney outer medulla) as ouabain (1.9 nmol/mg), with approximately the same affinity (Kk(ouabain)/Kd(NAB-ouabain) congruent to 1.6), and ouabain was fully competitive uith NAB-ouabain at these sites. NAB-ouabain binding and inhibition were reversible in the dark, but on exposure to ultraviolet light (310-370 nm) 30-40% of the binding and ihibition became irreversible; this binding was shown to be covalent by stability to trichloroacetic acid, organic solvents, and heat denaturation. Covalent labeling was prevented by photolysis of NAB-ouabain prior to the experiment, or by prior incubation of the enzyme with ouabain. On sodium dodecyl suffate-polyacrylamide gels of labeled Na,K-ATPase, about half of the covalently bound [3H]NAB-ouabain migrated with the large polypeptide (molecular weight congruent to 95 000), and half migrated with a small polypeptide (molecular weight congruent to 12 000); noncovalently bound NAB-ouabain (60-70% of total label) ran with the tracking dye. A similar labeling pattern was obtained utilizing NaI microsomes prepared from pig kidney outer medulla. The small polypeptide was characterized as an acidic proteolipid by extractability into acid chloroform/methanol; labeling of this component by NAB-ouabain is the first demonstration that it is directly associated with the Na,K-ATPase. The results of our characterization of NAB-ouabain show that it has the required specificity, covalency, and efficiency of labeling for application in structural studies of Na,K-ATPase subunit interactions.

Percolation-controlled conduction in chalcogenide glass semiconductors

The applicability of effective-medium percolation theory (EMPT) to the composition dependence of the electrical conductivity is discussed for binary and multicomponent chalcogenide glasses. Calculations assuming a mixture of atoms, which are applied to explain the experiment for As-Te films by Ast, do not agree with experiments on all the systems considered. By assuming a chemical which forms microscopic molecular species, the conductivity over the measured compositional range can be well described with EMPT in the As-Se, As-S, and ${\mathrm{As}}_{2}$${\mathrm{Se}}_{3}$-${\mathrm{Sb}}_{2}$${\mathrm{Se}}_{3}$ systems, except for Te-Se, ${\mathrm{As}}_{2}$${\mathrm{Se}}_{3}$-${\mathrm{As}}_{2}$${\mathrm{Te}}_{3}$, and ${\mathrm{As}}_{2}$${\mathrm{S}}_{2.63}$-Te which contain the isomorphous Te, Se, and S atoms. Chalcogenide glasses can be classified roughly into three groups: a random mixture of atoms type, a strong chemical ordering type, and an isomorphous-atom type. It is suggested that EMPT has broad application in structural studies of chalcogenide glasses.

Separation of Saccharomyces cerevisiae tRNAs on Two‐Dimensional Polyacrylamide Gels as Applied to Investigations on the Mutational Alterations of tRNA that Produce Nonsense Suppressors

Nonsense suppressors of the yeast Saccharomyces cerevisiae have been found that cause the translational suppression of UAA (ochre) or UAG (amber) nonsense mutants of iso‐1‐cytochrome c by inserting tyrosine, serine or leucine at the site of the nonsense codon. This report describes the fractionation of tyrosine, serine and leucine tRNAs from yeast on two‐dimensional polyacrylamide gels, as well as a partial characterisation of these species. There is only one tyrosine tRNA in yeast, whilst the serine and leucine tRNAs identified in this study can between them decode all the serine and leucine codons which differ in only one base from either UAA or UAG. We did not characterise those serine and leucine tRNAs whose cognate codons differ in more than one base from the amber or ochre codon.Separation of the tRNA of individual yeast strains on two‐dimensional gels can be used in conjunction with RNA fingerprinting techniques to investigate whether the amber or ochre suppressor mutations of yeast determine structural changes in the tyrosine, serine or leucine tRNAs. This approach has already allowed an elucidation of the tRNATyr structural change determined by tyrosine‐inserting amber suppressor mutations at the SUP5 locus. Although it has, to date, only been successfully applied to the sequencing of yeast amber suppressors, it has also a potential application in structural studies of ochre suppressors. One of the leucine tRNAs analysed in this study contains an unidentified modified nucleotide in its anticodon, a minor component of interest as it may also be present in the anticodon of yeast ochre suppressor tRNAs and cause the restrictive recognition of UAA but not UAG codons. The possible anticodon structures of yeast ochre‐suppressing tRNAs are discussed in the light of present knowledge of codon‐anticodon pairing.