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
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
Glycosylation not
only impacts the functions of glycoproteins but
can also improve glycoprotein stability and folding efficiencyî—¸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
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
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
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.
<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
<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
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
Workflow for the design of novel hDHODH inhibitors.
<p>Workflow for the design of novel hDHODH inhibitors.</p
The novel hDHODH inhibitors were constrained by steric contours in PhSIA.
<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