Interaction of Biliverdin Chromophore with Near-Infrared Fluorescent Protein BphP1-FP Engineered from Bacterial Phytochrome

Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.Peer reviewe

Ensuring the national security of agriculture in the digital era through the formation of human capital

Purpose: Over the past few years in many literary sources and the media an issue of the digital economy is rapidly gaining popularity due to the qualitative technological changes in society. In current circumstances the qualitative component of labor resources called “human capital” is a priority value. Design/Methodology/Approach: The following research methods have been used in the work: monographic, abstract-logical, sociological, economic-mathematical and calculation-constructive ones. Findings: The solution of the problem of ensuring food security of the population of the region depends on the methods and strategies of applying technologies in domestic and global agriculture, as well as on the correct and effective use of scientific-technical implementations in the industry. Practical implications: Identification of segments in tourism is mainly made based on their geographical, demographical, socio-economic, psychographic, behavior characteristics or their combinations. On the other hand, the authors stress on the fact that there is no universal segmentation approach and therefore, potential tourists can be classified based on other characteristics. Originality/Value: The use of digital technologies in the agricultural industry will increase the efficiency of production activities.peer-reviewe

Photophysical Properties of Fluorescent Probe Thioflavin T in Crowded Milieu

Thioflavin T (ThT) is a widely used fluorescent probe of amyloid fibrils, which accompanies many serious neurodegenerative and other diseases. Until recently, examinations of processes of amyloid fibril formation in vitro were conducted in solutions whose properties were significantly different from those found inside the densely packed cells. Such crowded cellular milieu is typically simulated in vitro using concentrated solutions of inert polymers, which do not usually interact with proteins. However, these crowding agents can have a direct effect on the ThT molecule, and this effect must be taken into account. We examined the influence of PEG-400, PEG-12000, and Dextran-70 on the photophysical properties of ThT. It was shown that these crowding agents caused the red shift of the absorption, fluorescence excitation, and fluorescence spectra of ThT. Under these conditions, the increases of the molar extinction coefficient, fluorescence quantum yield, and excitation lifetime of ThT are also observed. However, these changes are significantly less pronounced than those observed for ThT bound to fibrils. It is concluded that, despite some effects of crowding agents on intrinsic fluorescent properties of ThT, this dye can be used as a probe of structure and formation of amyloid fibrils in crowded milieu in vitro

Allosteric effects of chromophore interaction with dimeric near-infrared fluorescent proteins engineered from bacterial phytochromes

Fluorescent proteins (FPs) engineered from bacterial phytochromes attract attention as probes for in vivo imaging due to their near-infrared (NIR) spectra and use of available in mammalian cells biliverdin (BV) as chromophore. We studied spectral properties of the iRFP670, iRFP682 and iRFP713 proteins and their mutants having Cys residues able to bind BV either in both PAS (Cys15) and GAF (Cys256) domains, in one of these domains, or without these Cys residues. We show that the absorption and fluorescence spectra and the chromophore binding depend on the location of the Cys residues. Compared with NIR FPs in which BV covalently binds to Cys15 or those that incorporate BV noncovalently, the proteins with BV covalently bound to Cys256 have blue-shifted spectra and higher quantum yield. In dimeric NIR FPs without Cys15, the covalent binding of BV to Cys256 in one monomer allosterically inhibits the covalent binding of BV to the other monomer, whereas the presence of Cys15 allosterically promotes BV binding to Cys256 in both monomers. The NIR FPs with both Cys residues have the narrowest blue-shifted spectra and the highest quantum yield. Our analysis resulted in the iRFP713/Val256Cys protein with the highest brightness in mammalian cells among available NIR FPs.Peer reviewe

The unfolding of iRFP713 in a crowded milieu

The exploring of biological processes in vitro under conditions of macromolecular crowding is a way to achieve an understanding of how these processes occur in vivo. In this work, we study the unfolding of the fluorescent probe iRFP713 in crowded environment in vitro. Previously, we showed that the unfolding of the dimeric iRFP713 is accompanied by the formation of a compact monomer and an intermediate state of the protein. In the intermediate state, the macromolecules of iRFP713 have hydrophobic clusters exposed to the surface of the protein and are prone to aggregation. Concentrated solutions of polyethylene glycol (PEG-8000), Dextran-40 and Dextran-70 with a molecular mass of 8000, 40000 and 70000 Da, respectively, were used to model the conditions for macromolecular crowding. A limited available space provided by all the crowding agents used favors to the enhanced aggregation of iRFP713 in the intermediate state at the concentration of guanidine hydrochloride (GdnHCl), at which the charge of protein surface is neutralized by the guanidine cations. This is in line with the theory of the excluded volume. In concentrated solutions of the crowding agents (240–300 mg/ml), the stabilization of the structure of iRFP713 in the intermediate state is observed. PEG-8000 also enhances the stability of iRFP713 in the monomeric compact state, whereas in concentrated solutions of Dextran-40 and Dextran-70 the resistance of the protein in the monomeric state against GdnHCl-induced unfolding decreases. The obtained data argues for the excluded volume effect being not the only factor that contributes the behavior of biological molecules in a crowded milieu. Crowding agents do not affect the structure of the native dimer of iRFP713, which excludes the direct interactions between the target protein and the crowding agents. PEGs of different molecular mass and Dextran-40/Dextran-70 are known to influence the solvent properties of water. The solvent dipolarity/polarizability and basicity/acidity in aqueous solutions of these crowding agents vary in different ways. The change of the solvent properties in aqueous solutions of crowding agents might impact the functioning of a target protein

Four new species from the diatom (Bacillariophyceae) genus Adlafia Moser, Lange-Bertalot & Metzeltin from waterbodies of Vietnam

Four species of the diatom genus were found from waterbodies of Vietnam and described as new to science. Their formal descriptions are presented herein and they are illustrated by light and scanning electron micrographs. These new species are: Glushch., Kulik. Kociolek, , Glushch., Kulik. Kociolek, , Glushch., Kulik. Kociolek, and Glushch., Kulik. Kociolek, These species are then compared to other similar taxa. Our new findings add to the number of species in this interesting genus and contribute to our understanding of the unique diatom flora found in Vietnam. Keywords: , diatoms, morphology, new species, Southeast Asia, Vietnam</p

Two new species of Navicula (Bacillariophyta) from Southeast Asia

We present light and scanning electron microscopical observations on two new species of Navicula Bory sensu stricto from Southeast Asia. Navicula winoniformis Chudaev, Glushchenko, Kulikovskiy &amp; Kociolek, sp. nov. differs from taxa with similar outline and size by the combination of simple drop-like external raphe endings deflected to the primary valve side, presence of well-developed external longitudinal grooves and relatively low lineolae density. Navicula sparsilineolata Chudaev, Glushchenko, Kulikovskiy &amp; Kociolek, sp. nov. can be discriminated from the taxa of similar valve shape and size by consistently lower lineolae density, and from the majority of them also by the structure of external proximal raphe endings with small projections in proximal parts and larger triangular insertions in distal parts. Some remarks on Navicula species diversity and its distribution in the Southeast Asia are given. &nbsp;</p

Sensitivity of Superfolder GFP to Ionic Agents

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