Circular dichroism (CD) spectroscopy is one of the most versatile tools to study protein folding and to validate the proper fold of purified proteins. Here, we aim to provide a readily accessible, ...user-friendly and platform-independent tool capable of analysing multiple CD datasets of virtually any format and returning results as high-quality graphical output to the user.
CAPITO (CD Anaylsis and Plotting Tool) is a novel web server-based tool for analysing and plotting CD data. It allows reliable estimation of secondary structure content utilizing different approaches. CAPITO accepts multiple CD datasets and, hence, is well suited for a wide application range such as the analysis of temperature or pH-dependent (un)folding and the comparison of mutants.
http://capito.nmr.fli-leibniz.de.
cwiede@fli-leibniz.de or mago@fli-leibniz.de
Supplementary data are available at Bioinformatics online.
Sphingolipids (SL) represent a structurally diverse class of lipids that are central to cellular physiology and neuronal development and function. Defects in the sphingolipid metabolism are typically ...associated with nervous system disorders. The C4-dihydroceramide desaturase (DEGS1) catalyzes the conversion of dihydroceramide to ceramide, the final step in the SL de-novo synthesis. Loss of function mutations in DEGS1 cause a hypomyelinating leukodystrophy, which is associated with increased plasma dihydrosphingolipids (dhSL) and with the formation of an atypical SPB 18:1(14Z);O2 metabolite. Here, we characterize two novel DEGS1 variants of unknown significance (VUS), provide a structural model with a predicted substrate binding site and propose a regulatory link between DEGS1 and fatty acid desaturase 3 (FADS3). Both VUS involve single amino acid substitutions near the C-terminus within conserved regions of the enzyme. Patient 1 (p.R311K variant) shows severe progressive tetraspasticity, intellectual disability, and epilepsy in combination with brain magnetic resonance imaging (MRI) findings, typical for DEGS1-related leukodystrophy. Patient 2 (p.G270E variant) presents with delayed psychomotor development, oculomotor apraxia, and a normal brain MRI. Plasma from the p.R311K carrier showed a significantly elevated dhSL species and the presence of SPB 18:1(14Z);O2, while the plasma SL profile for the p.G270E variant was not altered. This suggests the p.R331K variant is pathogenic, while the p.G270E appears benign. As an increase in dihydroSL species is also seen in other pathological disorders of the SL metabolism, the SPB 18:1(14Z);O2 seems to be a more specific biomarker to discriminate between pathogenic and benign DEGS1 variants.
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The human organic cation transporter 2 (OCT2) is involved in the transport of endogenous quaternary amines and positively charged drugs across the basolateral membrane of proximal tubular cells. In ...the absence of a structure, the progress in unraveling the molecular basis of OCT2 substrate specificity is hampered by the unique complexity of OCT2 binding pocket, which seemingly contains multiple allosteric binding sites for different substrates. Here, we used the thermal shift assay (TSA) to better understand the thermodynamics governing OCT2 binding to different ligands.
Molecular modelling and
docking of different ligands revealed two distinct binding sites at OCT2 outer part of the cleft. The predicted interactions were assessed by
-inhibition assay using
H1-methyl-4-phenylpyridinium (
HMPP
) as a model substrate, or by measuring the uptake of radiolabeled ligands in intact cells. Crude membranes from HEK293 cells harboring human OCT2 (OCT2-HEK293) were solubilized in n-Dodecyl-β-D-Maltopyranoside (DDM), incubated with the ligand, heated over a temperature gradient, and then pelleted to remove heat-induced aggregates. The OCT2 in the supernatant was detected by western blot.
Among the compounds tested,
-inhibition and TSA assays showed partly overlapping results. Gentamicin and methotrexate (MTX) did not inhibit
HMPP
uptake but significantly increased the thermal stabilization of OCT2. Conversely, amiloride completely inhibited
HMPP
uptake but did not affect OCT2 thermal stabilization.
HMTX intracellular level was significantly higher in OCT2-HEK293 cells than in wild type cells. The magnitude of the thermal shift (ΔT
) did not provide information on the binding. Ligands with similar affinity showed markedly different ΔT
, indicating different enthalpic and entropic contributions for similar binding affinities. The ΔT
positively correlated with ligand molecular weight/chemical complexity, which typically has high entropic costs, suggesting that large ΔT
reflect a larger displacement of bound water molecules.
In conclusion, TSA might represent a viable approach to expand our knowledge on OCT2 binding descriptors.
Maturity stage affects the bioactive compounds as well as the antioxidant capacity in the fruit. This study was designed to identify and quantify carotenoids, as well as to evaluate vitamin E, ...vitamin C, antioxidant capacity and total phenolic compounds of
hips at different degrees of ripeness. HPLC (high performance liquid chromatography) analysis showed different types of carotenoids at different stages of maturity of
hips with significant differences (
˂ 0.05), where the maximum concentration was observed at late harvesting. In the hips investigated, only α-tocopherol was detected, the maximum concentration of both vitamin E and vitamin C was obtained in the orange hips with significant difference (
˂ 0.05). On the other hand, the highest hydrophilic and lipophilic TEAC (Trolox equivalent antioxidant capacity) values, as well as total phenolic contents, were determined in the mature hips (red colour) with significant difference (
< 0.0001) and (
< 0.001) respectively, whereas ORAC (oxygen radical absorbance capacity) showed lower activity in the mature hips with significant difference (
˂ 0.05). Late harvesting is recommended if a high content of carotenoids is desired, while harvesting should be carried out earlier if a higher vitamin E and vitamin C content is desired, which in turn affects the antioxidants capacity.
Teucrium hyrcanicum L. (family Lamiaceae) is widely distributed in the North and Northwest of Iran. It has been used in the form of tea, tonic, and tincture for the treatment of various diseases such ...as cough, rheumatism, and fever.
In this study, the total phenolic and flavonoid contents, antioxidant and cytotoxic activities of methanol extract and different fractions of T. hyrcanicum were measured. Furthermore, the potential ability of T. hyrcanicum to protect against H
O
-induced oxidative stress was tested on the NIH3T3 cell line. Then, the isolation and structure elucidation of the compounds were performed on the most potent fractions. Finally, the quantification of isolated compounds in methanol extract (ME) was done by the HPLC method. Isolated phytochemicals were assessed for the cytotoxic and antioxidant activities.
The results indicated that the methanol fraction (MF) had the highest amount of phenolic and flavonoid contents (69.36 mg GAE/g extract and 68.95 mg QE/g extract). The highest radical scavenging activities were observed from MF and ME (IC
44.32 and 61.12 μg.ml
, respectively). The best cytotoxicity was obtained by ethyl acetate fraction (EF) against A431 and MCF7 cell lines (IC
values of 235.4and 326.6 μg.ml
, respectively). The pretreatment with MF exerts the highest reduction in malondialdehyde (MDA) formation (IC
2.51 μM, p < 0.001) compared to the H
O
group (5.77 μM). Also, MF significantly inhibited H
O
-induced Glutathione (GSH) oxidation (p < 0.001). Furthermore, two phenolic compounds, acteoside and quercetin, were isolated and identified in MF and EF, respectively. The IC
values of acteoside and quercetin in the DPPH assay were 7.19 and 5.56 µg.ml
, respectively. Both quercetin and acteoside significantly reduced the MDA formation and inhibited GSH oxidation, which was comparable with BHA (as a standard antioxidant) (p < 0.05). Acteoside demonstrated significant cytotoxicity against all tested cell lines (IC
= 32 to 145 μg.ml
). The HPLC quantification of isolated compounds revealed that the quantity of acteoside and quercetin in ME were 93.31 and 16.87 μg.mg
, respectively.
The isolated compounds (quercetin and acteoside) had significant antioxidant activities and revealed a protective effect on H
O
-induced oxidative stress which was comparable with BHA.
Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium,
Chloracidobacterium
(
Cab
....)
thermophilum
, by
15
N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of
Cab. thermophilum
, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll
a
(BChl
a
), chlorophyll
a
(Chl
a
), and Zn-bacteriochlorophyll
a
′ (Zn-BChl
a
′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical
15
N NMR data, we assign the observed signals to a Chl
a
cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl
a
is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl
a
′. Chl
a
and 8
1
-OH Chl
a
have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl
a
molecule serves this role in all known homodimeric type-1 RCs.
NMR-based structure determination of a protein requires the assignment of resonances as indispensable first step. Even though heteronuclear through-bond correlation methods are available for that ...purpose, challenging situations arise in cases where the protein in question only yields samples of limited concentration and/or stability. Here we present a strategy based upon specific individual unlabeling of all 20 standard amino acids to complement standard NMR experiments and to achieve unambiguous backbone assignments for the fast precipitating 23 kDa catalytic domain of human aprataxin of which only incomplete standard NMR data sets could be obtained. Together with the validation of this approach utilizing the protein GB1 as a model, a comprehensive insight into metabolic interconversion ("scrambling”) of NH and CO groups in a standard
Escherichia coli
expression host is provided.
Abstract
Diatoms are abundant unicellular microalgae, responsible for ≈20 % of global photosynthetic CO
2
fixation. Nevertheless, we know little about fundamental aspects of their biology, such as ...their sexual reproduction. Pheromone‐mediated chemical communication is crucial for successful mating. An attraction pheromone was identified in the diatom
Seminavis robusta
, but metabolites priming cells for sex and synchronizing search and mating behavior remained elusive. These sex‐inducing pheromones (SIP) induce cell cycle arrest and trigger the production of the attraction pheromone. Here we describe the challenging structure elucidation of an
S. robusta
SIP. Guided by metabolomics, a candidate metabolite was identified and elucidated by labeling experiments, NMR, ESI MS
n
analyses, and chemical transformations. The use of negative ion mode MS was essential to decipher the unprecedented hydroxyproline and β‐sulfated aspartate‐containing cyclic heptapeptide that acts in femtomolar concentrations.
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•Band-selective 13Cα-15N het-TOCSY mixing can be an effective module in RF pulse schemes for resonance assignments in IDPs.•One-shot correlation spectra via simultaneous 15Nx and ...13Cαx magnetisation transfer from residue i to i+/-1 may be obtained.•The method may be modified to collect RNNCA HEHAHANH and R’CACAN HEHAHA(CACO)NH data (with R = ’HN’ or ’HNCO’ and R’ = ’Hα’ or sidechain ’HscCsc’).
Intrinsically disordered proteins (IDPs) or protein regions represent functionally important biomolecules without unique structure. Their inherent flexibility prevents high-resolution structure determination by X-ray or cryo-EM methods. In contrast, NMR spectroscopy provides an extensive and still growing set of experimental approaches to obtain detailed information on structure and dynamics of IDPs. Here, it is experimentally demonstrated that 15N-13Cα band-selective heteronuclear cross-polarisation that has been successfully employed recently to achieve the efficient transfer of 15Nx magnetisation from amino acid residue ’i’ to ’i + 1’ and ’i − 1’ residues in uniformly (15N,13C)-labelled intrinsically disordered proteins can also be applied to transfer, without significant relaxation losses, 13Cαx magnetisation from an amino acid residue to its neighbouring residues. The possibility to obtain in one-shot correlation spectra arising from the simultaneous transfer of 15Nx and 13Cαx magnetisations from an amino acid residue to neighbouring residues is also demonstrated.
Reactive polymersomes represent a versatile artificial cargo carrier system that can facilitate an immediate release in response to a specific stimulus. The herein presented oxidation‐sensitive ...polymersomes feature a time‐delayed release mechanism in an oxidative environment, which can be precisely adjusted by either tuning the membrane thickness or partial pre‐oxidation. These polymeric vesicles are conveniently prepared by PISA allowing the straightforward and effective in situ encapsulation of cargo molecules, as shown for dyes and enzymes. Kinetic studies revealed a critical degree of oxidation causing the destabilization of the membrane, while no release of the cargo is observed beforehand. The encapsulation of glucose oxidase directly transforms these polymersomes into glucose‐sensitive vesicles, as small molecules including sugars can passively penetrate their membrane. Considering the ease of preparation, these polymersomes represent a versatile platform for the confinement and burst release of cargo molecules after a precisely adjustable time span in the presence of specific triggers, such as H2O2 or glucose.
Polymersomes made by PISA based on thioether‐functionalized polymers as membrane‐forming block show a spontaneous membrane destabilization once a critical degree of oxidation is reached. Fine‐tuning of the degradation time can be performed either by adjusting the membrane thickness or by partial pre‐oxidation and can be further exploited to tailor the burst release of encapsulated dyes or enzymes in the presence of specific triggers.
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