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

Electron paramagnetic resonance studies of VO<SUP>2+</SUP> in single crystals of NaCl, KCl and RbCl

Electron paramagnetic resonance (EPR) of VO2+ radicals incorporated in face-centred single crystals of NaCl, KCl and RbCl has been studied in the temperature range of 77&#176; K to 330&#176; K. At liquid nitrogen temperature, anisotropic spectra have been recorded in each case while spectra recorded at room and higher temperatures are isotropic suggesting the existence of a fast readjustment of VO2+ molecular ions in the crystals at higher temperature while this motion gets hindered at liquid nitrogen temperatures. Spin-Hamiltonian constants are calculated from the recorded isotropic and anisotropic spectra. The line widths in each case are found to obey a parabolic law originally proposed by Kivelson. The constants which give a close fit have been evaluated. The random orientation and readjustment of V-0 bond in these alkali chloride crystals is explained on the assumption that the medium exhibits a "liquid-like" nature for VO2+ molecular ion as far as the electron paramagnetic resonance absorption is concerned

Intermediate obtained from photoionization, serving as precursor for the synthesis of Schiff’s base

In this article, we have introduced an intermediate benzyl carbocation (formed as a result of photoionization) which serves as precursor for the synthesis of Schiff’s base. Lifetimes of many carbocations were determined from our laboratory. During the determination of the lifetimes, our endeavor was to obtain a carbocation with high selectivity, s = knu/kH2O. The selectivity is the ratio of the rate constant of the reaction of carbocation with an externally added nucleophile, (nu, aniline) to that of the rate constant of the water. Our intention was to obtain a carbocation with high selectivity, so that one can pave a path for the synthesis of Schiff’s base by the reaction of the carbocation intermediate with aniline. DOI: http://dx.doi.org/10.4314/bcse.v28i2.1

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 &#948;-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, &#948;-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