Bacterial lipid modification of proteins for novel protein engineering applications

Functioning of proteins efficiently at the solid-liquid interface is critical to not only biological but also modern man-made systems such as ELISA, liposomes and biosensors. Anchoring hydrophilic proteins poses a major challenge in this regard. Lipid modification, N-acyl-S-diacylglyceryl-Cys, providing an N-terminal hydrophobic membrane anchor is a viable solution that bacteria have successfully evolved but remains unexploited. Based on the current understanding of this ubiquitous and unique bacterial lipid modification it is possible to use Escherichia coli, the popular recombinant protein expression host, for converting a non-lipoprotein to a lipoprotein with a hydrophobic anchor at the N-terminal end. We report two strategies applicable to non-lipoproteins (with or without signal sequences) employing minimal sequence change. Taking periplasmic Shigella apyrase as an example, its signal sequence was engineered to include a lipobox, an essential determinant for lipid modification, or its mature sequence was fused to the signal sequence of abundant outer membrane lipoprotein, Lpp. Lipid modification was proved by membrane localization, electrophoretic mobility shift and mass spectrometric analysis. Substrate specificity and specific activity measurements indicated functional integrity after modification. In conclusion, a convenient protein engineering strategy for converting non-lipoprotein to lipoprotein for commercial application has been devised and tested successfully

Studies on the synthesis of the toxins, pardaxin, δ-toxin and their analogues by solid-phase methods

Studies in our laboratory have been directed towards understanding the mechanism of action of two hydrophobic toxins, pardaxin comprising 33 residues and δ-toxin comprising 26 residues. Since isolation of these peptides in large amounts from natural sources is not convenient, we have explored synthetic approaches to get these peptides as well as their analogs. We have used chemistry specific to fluorenylmethoxycarbonyl (Fmoc) andt-butyloxycarbonyl (Boc) amino acids. Synthesis specific for Fmoc amino acids was carried out manually as well as on a semi-automated continuous flow peptide synthesizer. Synthesis specific for Boc amino acids was carried out manually. The protocols used by us have yielded 15-33 residue peptides which are of high purity. Even in peptides where heterogeneity was present, pure peptide could be obtained in good yields using simple gradients in fast performance liquid chromatography. The synthesis of pardaxin, δ-toxin and several analogs should help in identifying the molecular determinants of biological activity

EVpedia: a community web portal for extracellular vesicles research

Motivation: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. Results: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research.X1110478Ysciescopu

Кераміка «terra sigillata» з с. Зимне на Волині

Стаття присвячена публікації чотирьох керамічних посудин типу «terra sigillata», знайдених на дні р. Луги у с. Зимне Володимир-Волинського району Волинської області. Попередній аналіз цих знахідок дозволяє віднести їх до Понтійського центру виробництва такого посуду. Вірогідним шляхом потрапляння цієї колекції на Волинь була готська експансія у Північне Причорномор’я

Conformation of gramicidin a in water: inference from analysis of hydrogen/deuterium exchange behavior by matrix assisted laser desorption ionization mass spectrometry

Gramicidin A (the major component of gramicidin D) is a highly hydrophobic peptide with very little solubility in water. Hence, the conformation of this peptide has been extensively investigated in organic solvents and model membranes, but not in water. The peptide adopts a β6.3-helical conformation in the monomeric and dimeric forms. We have investigated the conformation of gramicidin A in water by monitoring hydrogen-deuterium exchange by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry. Our results indicate that gramicidin A is monomeric and exists in a highly folded conformation. The metal ion bound forms are clearly discernible in the monomers. The presence of the dimeric form is not observed. It is unlikely this is due to the operating conditions or the method used, as both hetero- and homodimers in gramicidin D are detected when methanol is used as a solvent. The present study also establishes that the linear gramicidins retain a history of solvent environment when ions are generated by matrix-assisted laser desorption ionization and analyzed by time-of-flight

Antibacterial activities and conformations of bovine β-defensin BNBD-12 and analogs: structural and disulfide bridge requirements for activity

Structure and biological activities of synthetic peptides corresponding to bovine neutrophil β-defensin BNBD-12, GPLSC1GRNGGVC2IPIRC3 PVPMRQIGTC4 FGRPVKC5 C6RSW with disulfide connectivities C1-C5, C2-C4 and C3-C6 and its variants with one, two and three disulfide bridges have been investigated. Selective protection of cysteine thiols was necessary in the four and six cysteine containing peptides for the formation of disulfide connectivities as observed in BNBD-12. Circular dichroism (CD) spectra indicate that in aqueous medium, only a small fraction of molecules populate turn-like conformations. In the presence of micelles and lipid vesicles, the single, two and three disulfide containing peptides adopt β-hairpin or β-sheet structures. Antibacterial activity was observed for all the peptides, irrespective of the number of disulfide bridges or how they were connected. Our results suggest that a rigid β-sheet structure or the presence of three disulfide bridges does not appear to be stringent requirements for antibacterial activity in β-defensins