27 research outputs found

    Simultaneous Analysis of Six Polymethoxyflavones and Six 5‑Hydroxy-polymethoxyflavones by High Performance Liquid Chromatography Combined with Linear Ion Trap Mass Spectrometry

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    Polymethoxyflavones (PMFs) and monohydroxylated polymethoxyflavones (OH-PMFs) exist exclusively in the citrus genus, particularly in citrus peels. Currently, due to the broad application of PMFs and OH-PMFs in nutraceuticals, pharmaceuticals, and functional foods, their identification and quantification will be of great significance and the first criteria to meet. We have developed a validated method with high performance liquid chromatography coupled with linear ion trap mass spectrometry. The method was fully validated in linearity, precision, accuracy, and recovery. Six PMFs and their monohydroxyl counterparts, six 5-OH-PMFs, were simultaneous analyzed within 20 min for the first time. The LOD (limit of detection) and LOQ (limit of quantitation) were calculated as 0.02–0.23 and 0.05–0.76 μg/mL, respectively. The method was performed on the samples of acid treated citrus peel extracts. The citrus peel extracts with high content of PMFs and 5-OH PMFs may provide reliable and economical resources in biological activity studies and development of health beneficial products

    Predictions for α‑Helical Glycopeptide Design from Structural Bioinformatics Analysis

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    Glycosylation not only impacts the functions of glycoproteins but can also improve glycoprotein stability and folding efficiencycharacteristics that are desirable for protein engineering and therapeutic design. To further elucidate the effects of N-glycosylation on protein structure and to provide principles useful for the rational design of α-helical glycopeptides, we investigate stabilizing protein–sugar interactions in α-helical glycosylation sites using an integrated structural bioinformatics analysis and molecular dynamics simulation approach. We identify two glycan conformations with an Asn χ<sub>1</sub> of 180° or 300° that are amenable to α-helical structure in natural α-helical glycosylation sites in the Protein Data Bank. A combination of sterics and favorable intraglycopeptide enthalpy explains the existence of only these two conformations. Furthermore, we catalog all known protein–sugar interactions that utilize these conformational modes. The most common interactions involve either a Glu residue at the −4 position interacting with the GlcNAc whose Asn has χ<sub>1</sub> = 300° or a Glu residue at the +4 position interacting with the GlcNAc whose Asn has χ<sub>1</sub> = 180°. Via metadynamics simulations of model α-helical glycopeptides with each of these two interactions, we find that both interactions are stabilizing as a result of favorable electrostatic intraglycopeptide interactions. Thus, we suggest that incorporating a Glu at either the −4 or +4 position relative to an N-linked glycan may be a useful strategy for engineering stable α-helical glycoproteins

    Insights into How Cyclic Peptides Switch Conformations

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    Cyclic peptides have recently emerged as promising modulators of protein–protein interactions. However, it is currently highly difficult to predict the structures of cyclic peptides owing to their rugged conformational free energy landscape, which prevents sampling of all thermodynamically relevant conformations. In this article, we first investigate how a relatively flexible cyclic hexapeptide switches conformations. It is found that, although the circular geometry of small cyclic peptides of size 6–8 may require rare, coherent dihedral changes to sample a new conformation, the changes are rather local, involving simultaneous changes of ϕ<sub><i>i</i></sub> and ψ<sub><i>i</i></sub> or ψ<sub><i>i</i></sub> and ϕ<sub><i>i+</i>1</sub>. The understanding of how these cyclic peptides switch conformations enables the use of metadynamics simulations with reaction coordinates specifically targeting such coupled two-dihedral changes to effectively sample cyclic peptide conformational space

    Are Protein Force Fields Getting Better? A Systematic Benchmark on 524 Diverse NMR Measurements

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    Recent hardware and software advances have enabled simulation studies of protein systems on biophysically relevant time scales, often revealing the need for improved force fields. Although early force field development was limited by the lack of direct comparisons between simulation and experiment, recent work from several laboratories has demonstrated direct calculation of NMR observables from protein simulations. Here, we quantitatively evaluate 11 recent molecular dynamics force fields in combination with 5 solvent models against a suite of 524 chemical shift and <i>J</i> coupling (<sup>3</sup><i>JH</i><sub><i>N</i></sub><i>H</i><sub>α</sub>, <sup>3</sup><i>JH</i><sub><i>N</i></sub><i>C</i><sub>β</sub>, <sup>3</sup><i>JH</i><sub>α</sub><i>C</i>′, <sup>3</sup><i>JH</i><sub><i>N</i></sub><i>C</i>′, and <sup>3</sup><i>JH</i><sub>α</sub><i>N</i>) measurements on dipeptides, tripeptides, tetra-alanine, and ubiquitin. Of the force fields examined (ff96, ff99, ff03, ff03*, ff03w, ff99sb*, ff99sb-ildn, ff99sb-ildn-phi, ff99sb-ildn-NMR, CHARMM27, and OPLS-AA), two force fields (ff99sb-ildn-phi, ff99sb-ildn-NMR) combining recent side chain and backbone torsion modifications achieved high accuracy in our benchmark. For the two optimal force fields, the calculation error is comparable to the uncertainty in the experimental comparison. This observation suggests that extracting additional force field improvements from NMR data may require increased accuracy in <i>J</i> coupling and chemical shift prediction. To further investigate the limitations of current force fields, we also consider conformational populations of dipeptides, which were recently estimated using vibrational spectroscopy

    Are Protein Force Fields Getting Better? A Systematic Benchmark on 524 Diverse NMR Measurements

    No full text
    Recent hardware and software advances have enabled simulation studies of protein systems on biophysically relevant time scales, often revealing the need for improved force fields. Although early force field development was limited by the lack of direct comparisons between simulation and experiment, recent work from several laboratories has demonstrated direct calculation of NMR observables from protein simulations. Here, we quantitatively evaluate 11 recent molecular dynamics force fields in combination with 5 solvent models against a suite of 524 chemical shift and <i>J</i> coupling (<sup>3</sup><i>JH</i><sub><i>N</i></sub><i>H</i><sub>α</sub>, <sup>3</sup><i>JH</i><sub><i>N</i></sub><i>C</i><sub>β</sub>, <sup>3</sup><i>JH</i><sub>α</sub><i>C</i>′, <sup>3</sup><i>JH</i><sub><i>N</i></sub><i>C</i>′, and <sup>3</sup><i>JH</i><sub>α</sub><i>N</i>) measurements on dipeptides, tripeptides, tetra-alanine, and ubiquitin. Of the force fields examined (ff96, ff99, ff03, ff03*, ff03w, ff99sb*, ff99sb-ildn, ff99sb-ildn-phi, ff99sb-ildn-NMR, CHARMM27, and OPLS-AA), two force fields (ff99sb-ildn-phi, ff99sb-ildn-NMR) combining recent side chain and backbone torsion modifications achieved high accuracy in our benchmark. For the two optimal force fields, the calculation error is comparable to the uncertainty in the experimental comparison. This observation suggests that extracting additional force field improvements from NMR data may require increased accuracy in <i>J</i> coupling and chemical shift prediction. To further investigate the limitations of current force fields, we also consider conformational populations of dipeptides, which were recently estimated using vibrational spectroscopy

    Steric contour regions calculated using the PhSIA.

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    <p>(a) Inhibitor 1 (most active), and (<b>b</b>) inhibitor 25 (inactive). Steric allowed and disallowed regions are shown by green (contribution level, 80%) and yellow contours (contribution level, 20%), respectively.</p

    Development of a Human Dihydroorotate Dehydrogenase (hDHODH) Pharma-Similarity Index Approach with Scaffold-Hopping Strategy for the Design of Novel Potential Inhibitors

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    <div><p>Human dihydroorotate dehydrogenase (hDHODH) is a class-2 dihydroorotate dehydrogenase. Because it is extensively used by proliferating cells, its inhibition in autoimmune and inflammatory diseases, cancers, and multiple sclerosis is of substantial clinical importance. In this study, we had two aims. The first was to develop an hDHODH pharma-similarity index approach (PhSIA) using integrated molecular dynamics calculations, pharmacophore hypothesis, and comparative molecular similarity index analysis (CoMSIA) contour information techniques. The approach, for the discovery and design of novel inhibitors, was based on 25 diverse known hDHODH inhibitors. Three statistical methods were used to verify the performance of hDHODH PhSIA. Fischer’s cross-validation test provided a 98% confidence level and the goodness of hit (GH) test score was 0.61. The <i>q<sup>2</sup></i>, <i>r<sup>2</sup></i>, and predictive <i>r<sup>2</sup></i> values were 0.55, 0.97, and 0.92, respectively, for a partial least squares validation method. In our approach, each diverse inhibitor structure could easily be aligned with contour information, and common substructures were unnecessary. For our second aim, we used the proposed approach to design 13 novel hDHODH inhibitors using a scaffold-hopping strategy. Chemical features of the approach were divided into two groups, and the Vitas-M Laboratory fragment was used to create de novo inhibitors. This approach provides a useful tool for the discovery and design of potential inhibitors of hDHODH, and does not require docking analysis; thus, our method can assist medicinal chemists in their efforts to identify novel inhibitors.</p></div

    A Perfluoroaryl-Cysteine S<sub>N</sub>Ar Chemistry Approach to Unprotected Peptide Stapling

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    We report the discovery of a facile transformation between perfluoroaromatic molecules and a cysteine thiolate, which is arylated at room temperature. This new approach enabled us to selectively modify cysteine residues in unprotected peptides, providing access to variants containing rigid perfluoroaromatic staples. This stapling modification performed on a peptide sequence designed to bind the C-terminal domain of an HIV-1 capsid assembly polyprotein (C-CA) showed enhancement in binding, cell permeability, and proteolytic stability properties, as compared to the unstapled analog. Importantly, chemical stability of the formed staples allowed us to use this motif in the native chemical ligation-mediated synthesis of a small protein affibody that is capable of binding the human epidermal growth factor 2 receptor

    The novel hDHODH inhibitors were constrained by steric contours in PhSIA.

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    <p><b>(a)</b> The novel hDHODH inhibitor (EnFrag01), predicted to highly active. (<b>b</b>) The novel hDHODH inhibitor (EnFrag13), predicted to be inactivity.</p
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