Structures of viscotoxins A1 and B2 from European mistletoe solved using native data alone.
ABSTRACT Crystals of the cytotoxic thionin proteins viscotoxins A1 and B2 extracted from mistletoe diffracted to high resolution (1.25 and 1.05 A, respectively) and are excellent candidates for testing crystallographic methods. Ab initio direct methods were only successful in solving the viscotoxin B2 structure, which with 861 unique non-H atoms is one of the largest unknown structures without an atom heavier than sulfur to be solved in this way, but sulfur-SAD phasing provided a convincing solution for viscotoxin A1. Both proteins form dimers in the crystal and viscotoxin B2 (net charge +4 per monomer), but not viscotoxin A1 (net charge +6), is coordinated by sulfate or phosphate anions. The viscotoxin A1 crystal has a higher solvent content than the viscotoxin B2 crystal (49% as opposed to 28%) with solvent channels along the crystallographic 4(3) axes.
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ABSTRACT: Thionins are cysteine-rich, biologically active small (∼5 kDa) and basic proteins occurring ubiquitously in the plant kingdom. This study describes an efficient solid-phase extraction (SPE) method for the selective isolation of these pharmacologically active proteins. Hollow-monolithic extraction tips based on poly(styrene-co-divinylbenzene) with embedded zirconium silicate nano-powder were designed, which showed an excellent selectivity for sulphur-rich proteins owing to strong co-ordination between zirconium and the sulphur atoms from the thiol-group of cysteine. The sorbent provides a combination of strong hydrophobic and electrostatic interactions which may help in targeted separation of certain classes of proteins in a complex mixture based upon the binding strength of different proteins. European mistletoe, wheat and barley samples were used for selective isolation of viscotoxins, purothionins and hordothionins, respectively. The enriched fractions were subjected to analysis by matrix-assisted laser desorption/ionisation-time-of-flight mass spectrometer to prove the selectivity of the SPE method towards thionins. For peptide mass-fingerprint analysis, tryptic digests of SPE eluates were examined. Reversed-phase high-performance liquid chromatography hyphenated to diode-array detection was employed for the purification of individual isoforms. The developed method was found to be highly specific for the isolation and purification of thionins.Analytical and Bioanalytical Chemistry 07/2013; · 3.66 Impact Factor
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ABSTRACT: Thionins belong to a family of cysteine-rich, low-molecular-weight (∼5 KDa) biologically active proteins in the plant kingdom. They display a broad cellular toxicity against a wide range of organisms and eukaryotic cell lines. Thionins protect plants against different pathogens, including bacteria and fungi. A highly selective solid-phase extraction method for plant thionins is reported deploying aluminum silicate (3:2 mullite) powder as a sorbent in extraction columns. Mullite was shown to considerably improve selectivity compared to previously described zirconium silicate embedded poly(styrene-co-divinylbenzene) monolithic polymer. Due to the presence of aluminum(III), mullite offers electrostatic interactions for the selective isolation of cysteine rich proteins. In comparison to zirconium(IV) silicate, aluminum(III) silicate showed reduced interactions towards proteins which resulted into superior washings of unspecific compounds while still retaining cysteine rich thionins. In the presented study European mistletoe, wheat and barley samples were subjected to solid-phase extraction analysis for isolation of viscotoxins, purothionins and hordothionins, respectively. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for determining the selectivity of the sorbent towards thionins. The selectively retained thionins were quantified by colorimetric detection using the bicinchoninic acid assay. For peptide mass-fingerprint analysis tryptic digests of eluates were examined. This article is protected by copyright. All rights reserved.Journal of Separation Science 06/2014; · 2.59 Impact Factor
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ABSTRACT: De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that inhouse Zn-SAD phasing can be widely applicable to structure determination.Molecules and Cells 05/2013; · 2.21 Impact Factor