Chemical Shift Index Research Articles

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex invitro.

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

CSI 2.0: a significantly improved version of the Chemical Shift Index.

Protein chemical shifts have long been used by NMR spectroscopists to assist with secondary structure assignment and to provide useful distance and torsion angle constraint data for structure determination. One of the most widely used methods for secondary structure identification is called the Chemical Shift Index (CSI). The CSI method uses a simple digital chemical shift filter to locate secondary structures along the protein chain using backbone (13)C and (1)H chemical shifts. While the CSI method is simple to use and easy to implement, it is only about 75-80% accurate. Here we describe a significantly improved version of the CSI (2.0) that uses machine-learning techniques to combine all six backbone chemical shifts ((13)Cα, (13)Cβ, (13)C, (15)N, (1)HN, (1)Hα) with sequence-derived features to perform far more accurate secondary structure identification. Our tests indicate that CSI 2.0 achieved an average identification accuracy (Q3) of 90.56% for a training set of 181 proteins in a repeated tenfold cross-validation and 89.35% for a test set of 59 proteins. This represents a significant improvement over other state-of-the-art chemical shift-based methods. In particular, the level of performance of CSI 2.0 is equal to that of standard methods, such as DSSP and STRIDE, used to identify secondary structures via 3D coordinate data. This suggests that CSI 2.0 could be used both in providing accurate NMR constraint data in the early stages of protein structure determination as well as in defining secondary structure locations in the final protein model(s). A CSI 2.0 web server (http://csi.wishartlab.com) is available for submitting the input queries for secondary structure identification.

Enhanced Chemical Shift Analysis for Secondary Structure prediction of protein

Predicting secondary structure of protein through assigned backbone chemical shifts has been used widely because of its convenience and flexibility. In spite of its usefulness, chemical shift based analysis has some defects including isotopic shifts and solvent interaction. Here, it is shown that corrected chemical shift analysis for secondary structure of protein. It is included chemical shift correction through consideration of deuterium isotopic effect and calculate chemical shift index using probability-based methods. Enhanced method was applied successfully to one of the proteins from Mycobacterium tuberculosis. It is suggested that correction of chemical shift analysis could increase accuracy of secondary structure prediction of protein and small molecule in solution.

Theoretical study of the oxidation mechanisms of naphthalene initiated by hydroxyl radicals: the OH-addition pathway.

The oxidation mechanisms of naphthalene by OH radicals under inert (He) conditions have been studied using density functional theory along with various exchange-correlation functionals. Comparison has been made with benchmark CBS-QB3 theoretical results. Kinetic rate constants were correspondingly estimated by means of transition state theory and statistical Rice-Ramsperger-Kassel-Marcus (RRKM) theory. Comparison with experiment confirms that, on the OH-addition reaction pathway leading to 1-naphthol, the first bimolecular reaction step has an effective negative activation energy around -1.5 kcal mol(-1), whereas this step is characterized by an activation energy around 1 kcal mol(-1) on the OH-addition reaction pathway leading to 2-naphthol. Effective rate constants have been calculated according to a steady state analysis upon a two-step model reaction mechanism. In line with experiment, the correspondingly obtained branching ratios indicate that, at temperatures lower than 410 K, the most abundant product resulting from the oxidation of naphthalene by OH radicals must be 1-naphthol. The regioselectivity of the OH(•)-addition onto naphthalene decreases with increasing temperatures and decreasing pressures. Because of slightly positive or even negative activation energies, the RRKM calculations demonstrate that the transition state approximation breaks down at ambient pressure (1 bar) for the first bimolecular reaction steps. Overwhelmingly high pressures, larger than 10(5) bar, would be required for restoring to some extent (within ∼5% accuracy) the validity of this approximation for all the reaction channels that are involved in the OH-addition pathway. Analysis of the computed structures, bond orders, and free energy profiles demonstrate that all reaction steps involved in the oxidation of naphthalene by OH radicals satisfy Leffler-Hammond's principle. Nucleus independent chemical shift indices and natural bond orbital analysis also show that the computed activation and reaction energies are largely dictated by alterations of aromaticity, and, to a lesser extent, by anomeric and hyperconjugative effects.

Probing the U-Shaped Conformation of Caveolin-1 in a Bilayer

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82–S136; Cav182–136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics simulations. The secondary structure of Cav182–136 from NMR chemical shift indexing analysis serves as a guideline for generating initial structural models. Fifty independent molecular dynamics simulations (100 ns each) are performed to identify its favorable conformation and orientation in the bilayer. A representative configuration was chosen from these multiple simulations and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182–136 insertion depth in the bilayer), corroborate that Cav182–136 inserts in the membrane with U-shaped conformations, and show that the angle between H1 and H2 ranges from 35 to 69°, and the tilt angle of Cav182–136 is 27 ± 6°. The simulations also reveal that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.

Caveolin in Bilayers: Can the Intramembrane U-Shaped Conformation Really Exist

Caveolin induces membrane curvature and drives the formation of caveolae that participate in many crucial cell functions such as endocytosis. The central portion of caveolin-1 contains two helices (H1 and H2) connected by a three-residue break with both N- and C-termini exposed to the cytoplasm. Although a U-shaped configuration is assumed based on its inaccessibility by extracellular matrix probes, caveolin structure in a bilayer remains elusive. This work aims to characterize the structure and dynamics of caveolin-1 (D82 to S136; Cav182-136) in a DMPC bilayer using NMR, fluorescence emission measurements, and molecular dynamics (MD) simulations. The secondary structure of Cav182-136 from NMR chemical shift indexing analysis serves as guideline for generating initial structural models. 50 independent MD simulations (80 ns each) are performed to identify its favorable conformation and orientation in the bilayer. Using the short simulations as a guideline, a representative configuration was chosen and simulated for 1 μs to further explore its stability and dynamics. The results of these simulations mirror those from the tryptophan fluorescence measurements (i.e., Cav182-136 insertion depth in the bilayer), and corroborate that Cav182-136 inserts in the membrane with a U-shaped conformation. The angle between H1 and H2 ranges from 35° to 69°, and the tilt angle of Cav182-136 is 27° ± 6°. The simulations also show that specific faces of H1 and H2 prefer to interact with each other and with lipid molecules, and these interactions stabilize the U-shaped conformation.

A study on the aromaticity of [n]phenacenes and [n]helicenes (n = 3–9)

We studied the global and local aromaticity of [n]phenacenes and [n]helicenes for n=3–9 using the topological resonance energy and the magnetic resonance energy methods. The local aromaticity of the individual rings was studied using the bond resonance energy, the circuit resonance energy, and the ring current methods. Our results were compared with the results obtained by others who used the para-delocalization index, the harmonic oscillator model of aromaticity, and the nucleus-independent chemical shift indices. We found that sextet rings in the Clar structure are the main source of aromatic stabilization.

Fully and partially exohydrogenated Si80 fullerene cage: a DFT study

We have performed a density functional study to investigate electronic and magnetic properties of fully and partially exohydrogenation in the Si80 fullerene cage based on NMR parameters, nucleus-independent chemical shift (NICS) indices and natural population analysis. Hydrogenation of 20 silicons inside and 60 silicons outside results in two distinct values for the 29Si σ iso, and also for 29Si Δσ. NICS yields negative value of −1.5 ppm at the cage center of the fully exohydrogenated fullerene, Si80H80. However, NICS yields positive value of 1.5 ppm at the cage center of the partially exohydrogenated fullerene, H20@Si80H60, then becomes more negative along the axis toward the center of pentagon until reaching −3.0 ppm at the ring center (and −4.3 at the point 1 A away from the ring center outside), suggesting that in this case pentagons can be considered as aromatic fragments. H20@Si80H60 cage is composed of negatively charged hydrogen atoms and positively charged silicon atoms, indicating electron transfer from silicon atoms on the surfaces of the cage to the chemically bonded hydrogen atoms. A decreasing trend is observed for 1H σ iso values as the natural charges of hydrogen sites in these fullerenes increase while no regular trend is observed for the chemical shielding anisotropy Δσ of hydrogen atoms. .

The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids

Anionic and zwitterionic micelles are often used as simple models for the lipids found in bacterial and mammalian cell membranes to investigate antimicrobial peptide-lipid interactions. In our laboratory we have employed a variety of 1D, 2D, and diffusion ordered (DOSY) NMR experiments to investigate the interactions of antimicrobial peptides containing unnatural amino acids with SDS and DPC micelles. Complete assignment of the proton spectra of these peptides is prohibited by the incorporation of a high percentage of unnatural amino acids which don't contain amide protons into the backbone. However preliminary assignment of the TOCSY spectra of compound 23 in the presence of both micelles indicated multiple conformers are present as a result of binding to these micelles. Chemical Shift Indexing agreed with previously collected CD spectra that indicated on binding to SDS micelles compound 23 adopts a mixture of α-helical structures and on binding to DPC micelles this peptide adopts a mixture of helical and β-turn/sheet like structures. DOSY NMR experiments also indicated that the total positive charge and the relative placement of that charge at the N-terminus or C-terminus are important in determining the mole fraction of the peptide that will bind to the different micelles. DOSY and (1) H-NMR experiments indicated that the length of Spacer #1 plays a major role in defining the binding conformation of these analogs with SDS micelles. Results obtained from molecular simulations studies of the binding of compounds 23 and 36 with SDS micelles were consistent with the observed NMR results.

NMR Analysis of a Novel Enzymatically Active Unlinked Dengue NS2B-NS3 Protease Complex

The dengue virus (DENV) is a mosquito-borne pathogen responsible for an estimated 100 million human infections annually. The viral genome encodes a two-component trypsin-like protease that contains the cofactor region from the nonstructural protein NS2B and the protease domain from NS3 (NS3pro). The NS2B-NS3pro complex plays a crucial role in viral maturation and has been identified as a potential drug target. Using a DENV protease construct containing NS2B covalently linked to NS3pro via a Gly4-Ser-Gly4 linker ("linked protease"), previous x-ray crystal structures show that the C-terminal fragment of NS2B is remote from NS3pro and exists in an open state in the absence of an inhibitor; however, in the presence of an inhibitor, NS2B complexes with NS3pro to form a closed state. This linked enzyme produced NMR spectra with severe signal overlap and line broadening. To obtain a protease construct with a resolved NMR spectrum, we expressed and purified an unlinked protease complex containing a 50-residue segment of the NS2B cofactor region and NS3pro without the glycine linker using a coexpression system. This unlinked protease complex was catalytically active at neutral pH in the absence of glycerol and produced dispersed cross-peaks in a (1)H-(15)N heteronuclear single quantum correlation spectrum that enabled us to conduct backbone assignments using conventional techniques. In addition, titration with an active-site peptide aldehyde inhibitor and paramagnetic relaxation enhancement studies demonstrated that the unlinked DENV protease exists predominantly in a closed conformation in solution. This protease complex can serve as a useful tool for drug discovery against DENV.

Backbone and side-chain 1H, 13C and 15N assignments of the PPIase domain of macrophage infectivity potentiator (Mip) protein from Coxiella burnetii

Coxiella burnetii is an obligate intracellular gram-negative bacterium uniquely evolved to thrive in the inhospitable phagolysosome of macrophage. C. burnetii causes Q fever in humans and animals, which is emerging as a global public health concern. It is highly infectious and designated as a category B biowarfare agent because of its ubiquitous nature, abundant natural reservoirs, high resistance to environmental conditions, ease of transmission and low infectious dose. The lack of knowledge and awareness of C. burnetii leads to under-reporting and under-diagnosing of Q fever cases. Therefore, further understanding of the interactions between the infected host and the bacteria is necessary. C. burnetii macrophage infectivity potentiator (cb-Mip) is a secreted protein of 230 amino acids involving in intracellular survival of the pathogen. cb-Mip belongs to the family of FK506 binding protein, which possesses peptidyl-prolyl cis/trans isomerase (PPIase) activity. Besides acting as a PPIase, Mip protein homolog has been identified as virulence factor of many intracellular pathogenic microorganisms. In the present study, we report the near complete resonance assignments of the PPIase domain-containing region of Mip protein of C. burnetii. Secondary structure prediction based on chemical shift index analysis indicates that the protein adopts a predominately beta-strand structure, which is consistent with the crystal structure of homologous Mip protein in Legionella pneumophila.

Spectroscopic and theoretical studies on the aromaticity of pyrrol-2-yl-carbonyl conformers

The aromaticity of s-cis and s-trans pyrrol-2-yl carbonyl conformers was studied by FT-IR, 1H NMR spectroscopy and DFT calculations at the B3LYP/6-311++G(d,p) level of theory. The Harmonic Oscillator Model of Aromaticity (HOMA) and Nucleus Independent Chemical Shift (NICS) indices were calculated to estimate π-electron delocalization in the pyrrole ring. The usefulness of infrared spectroscopy in the evaluation of the aromaticity of the homogeneous set of pyrroles is discussed. The influence of 2-substitution on different aspects of aromaticity and stability of the pyrrol-2-yl carbonyl conformers is also discussed. It is concluded that the substitution effect of the title pyrrole derivatives can be explained on the basis of theoretical and experimental measurements of π-electron delocalization, including IR data.

CLUSTERGEN: a program for molecular cluster generation from crystallographic data

A new program,CLUSTERGEN, for molecular cluster generation is introduced.CLUSTERGENprovides the quantum mechanics/molecular mechanics (QM/MM) input files for program packages such asADF[Baerendset al.(2012). Vrije Universiteit, Amsterdam, The Netherlands] andGAUSSIAN[Frischet al.(2009). Gaussian Inc., Pittsburgh, Pennsylvania, USA]. Additionally, it prints out a standardCRYSTAL[Dovesiet al.(2009). University of Turin, Italy] input and, in general, facilitates file-format manipulation. TheCLUSTERGENprogram is supported by an extensive manual and a user-friendly graphical interface. The code is freely available and carefully commented, which makes it easily modifiable. Exemplary applications ofCLUSTERGENconcerning QM/MM calculations and derivation of nucleus-independent chemical shift indices are demonstrated.

A probabilistic model for secondary structure prediction from protein chemical shifts

Protein chemical shifts encode detailed structural information that is difficult and computationally costly to describe at a fundamental level. Statistical and machine learning approaches have been used to infer correlations between chemical shifts and secondary structure from experimental chemical shifts. These methods range from simple statistics such as the chemical shift index to complex methods using neural networks. Notwithstanding their higher accuracy, more complex approaches tend to obscure the relationship between secondary structure and chemical shift and often involve many parameters that need to be trained. We present hidden Markov models (HMMs) with Gaussian emission probabilities to model the dependence between protein chemical shifts and secondary structure. The continuous emission probabilities are modeled as conditional probabilities for a given amino acid and secondary structure type. Using these distributions as outputs of first- and second-order HMMs, we achieve a prediction accuracy of 82.3%, which is competitive with existing methods for predicting secondary structure from protein chemical shifts. Incorporation of sequence-based secondary structure prediction into our HMM improves the prediction accuracy to 84.0%. Our findings suggest that an HMM with correlated Gaussian distributions conditioned on the secondary structure provides an adequate generative model of chemical shifts.

MR imaging of renal cortical tumours: qualitative and quantitative chemical shift imaging parameters

To assess qualitative and quantitative chemical shift MRI parameters of renal cortical tumours. A total of 251 consecutive patients underwent 1.5-T MRI before nephrectomy. Two readers (R1, R2) independently evaluated all tumours visually for a decrease in signal intensity (SI) on opposed- compared with in-phase chemical shift images. In addition, SI was measured on in- and opposed-phase images (SI(IP), SI(OP)) and the chemical shift index was calculated as a measure of percentage SI change. Histopathology served as the standard of reference. A visual decrease in SI was identified significantly more often in clear cell renal cell carcinoma (RCCs) (R1, 73 %; R2, 64 %) and angiomyolipomas (both, 80 %) than in oncocytomas (29 %, 12 %), papillary (29 %, 17 %) and chromophobe RCCs (13 %, 9 %; all, P < 0.05). Median chemical shift index was significantly greater in clear cell RCC and angiomyolipoma than in the other histological subtypes (both, P < 0.001). Interobserver agreement was fair for visual (kappa, 0.4) and excellent for quantitative analysis (concordance correlation coefficient, 0.80). A decrease in SI on opposed-phase chemical shift images is not an identifying feature of clear cell RCCs or angiomyolipomas, but can also be observed in oncocytomas, papillary and chromophobe RCCs. After excluding angiomyolipomas, a decrease in SI of more than 25 % was diagnostic for clear cell RCCs. • Chemical shift MRI offers new information about fat within renal tumours. • Opposed-phase signal decrease can be observed in all renal cortical tumours. • A greater than 25 % decrease in signal appears to be diagnostic for clear cell RCCs.

1H, 13C and 15N resonance assignments of human parvulin 17

A 25-residue elongation at the N-terminus endows parvulin 17 (Par17) with altered functional properties compared to parvulin 14 (Par14), such as an enhanced influence on microtubule assembly. Therefore the three-dimensional structure of this N-terminal elongation is of particular interest. Here, we report the nearly complete (1)H, (13)C and (15)N chemical shift assignments of Par17. Subsequent chemical shift index analysis indicated that Par17 features a parvulin-type PPIase domain at the C-terminus, analogous to Par14, and an unstructured N-terminus encompassing the first 60 residues. Hence the N-terminus of Par17 apparently adopts a functionally-relevant structure only in presence of the respective interaction partner(s).

Solution Structure of a Phytocystatin from Ananas comosus and Its Molecular Interaction with Papain

The structure of a recombinant pineapple cystatin (AcCYS) was determined by NMR with the RMSD of backbone and heavy atoms of twenty lowest energy structures of 0.56 and 1.11 Å, respectively. It reveals an unstructured N-terminal extension and a compact inhibitory domain comprising a four-stranded antiparallel β-sheet wrapped around a central α-helix. The three structural motifs (G45, Q89XVXG, and W120) putatively responsible for the interaction with papain-like proteases are located in one side of AcCYS. Significant chemical shift perturbations in two loop regions, residues 45 to 48 (GIYD) and residues 89 to 91 (QVV), of AcCYS strongly suggest their involvement in the binding to papain, consistent with studies on other members of the cystatin family. However, the highly conserved W120 appears not to be involved in the binding with papain as no chemical shift perturbation was observed. Chemical shift index analysis further indicates that the length of the α-helix is shortened upon association with papain. Collectively, our data suggest that AcCYS undergoes local secondary structural rearrangements when papain is brought into close contact. A molecular model of AcCYS/papain complex is proposed to illustrate the interaction between AcCYS and papain, indicating a complete blockade of the catalytic triad by AcCYS.

Nucleus-independent chemical shift criterion for aromaticity in π-extended tetraoxa[8]circulenes

Recently synthesized π-extended symmetrical tetraoxa[8]circulenes that exhibit electroluminescent properties were calculated at the density functional theory (DFT) level using the quantum theory of atoms in molecules (QTAIM) approach to electron density distribution analysis. Nucleus-independent chemical shift (NICS) indices were used to characterize the aromaticity of the studied molecules. The tetraoxa[8]circulene molecules were found to consist of two antiaromatic perimeters (according to the Hückel "4n" antiaromaticity rule) that include 8 and 24 π-electrons. Conversely, NICS calculations demonstrated the existence of a common π-extended system (distributed like a flat ribbon) in the studied tetraoxa[8]circulene molecules. Thus, these symmetrical tetraoxa[8]circulene molecules provide examples of diatropic systems characterized by the presence of induced diatropic ring currents.

Resonance assignments of Ca2+-bound human S100A11

The S100 family belongs to the EF-hand calcium-binding proteins regulating a wide range of important cellular processes via protein-protein interactions. Most S100 proteins adopt a conformation of non-covalent homodimer for their functions. Calcium binding to the EF-hand motifs of S100 proteins is essential for triggering the structural changes, promoting exposure of hydrophobic regions necessary for target protein interactions. S100A11 is a protein found in diverse tissues and possesses multiple functions upon binding to different target proteins. RAGE is a multiligand receptor binding to S100A11 and the interactions at molecular level have not been reported. However, the three-dimensional structure of human S100A11 containing 105 amino acids is still not available for further interaction studies. To determine the solution structure, for the first time we report the (1)H, (15)N and (13)C resonance assignments and protein secondary structure prediction of human S100A11 dimer in complex with calcium using a variety of triple resonance NMR experiments and the chemical shift index (CSI) method, respectively.

Chemical shift assignments of the canecystatin-1 from Saccharum officinarum

Cystatins are cysteine proteases inhibitors that are widely distributed among insects, mammalians and plants. Here we report the complete resonance assignment of canecystatin-1 from Saccharum officinarum obtained by heteronuclear multidimensional high-resolution nuclear magnetic resonance spectroscopy. The consensus chemical shift index was calculated and showed the presence of one α-helix (residues 27-43) and three β-strands (residues 48-74, 78-89 and 94-104), a secondary structure pattern that suggests a domain-swapped structure as presented by stefin B and human cystatin C, opposed to the monomeric structure yet found in other phytocystatins like oryza and pineapple cystatin.