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

Temporal Correlations of Local Network Losses

We introduce a continuum model describing data losses in a single node of a packet-switched network (like the Internet) which preserves the discrete nature of the data loss process. {\em By construction}, the model has critical behavior with a sharp transition from exponentially small to finite losses with increasing data arrival rate. We show that such a model exhibits strong fluctuations in the loss rate at the critical point and non-Markovian power-law correlations in time, in spite of the Markovian character of the data arrival process. The continuum model allows for rather general incoming data packet distributions and can be naturally generalized to consider the buffer server idleness statistics

Stabilization of structure in near-infrared fluorescent proteins by binding of biliverdin chromophore

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes and their mutants with different location of Cys residues, which able to bind a biliverdin chromophore, or without these Cys residues were studied using intrinsic tryptophan fluorescence, NIR fluorescence and circular dichroism. It was shown that a covalent binding of the biliverdin chromophore to a Cys residue via thioether group substantially stabilizes the spatial structure of NIR FPs. The stability of the protein structure and the chromophore association strength strongly depends on the location of Cys residues and decreases in the following order: a protein with Cys residues in both domains, a protein with Cys in PAS domains, and a protein with Cys in GAF domains. NIR FPs without Cys residues capable to covalently attach biliverdin have the lowest stability, comparable to NIR FP apoforms. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

The higher-order magnetic skyrmions in non-uniform magnetic fields

For 2D Hubbard model with spin-orbit Rashba coupling in external magnetic field the structure of effective spin interactions is studied in the regime of strong electron correlations and at half-filling. It is shown that in the third order of perturbation theory, the scalar and vector chiral spin-spin interactions of the same order arise. The emergence of the latter is due to orbital effects of magnetic field. It is shown that for nonuniform fields, scalar chiral interaction can lead to stabilization of axially symmetric skyrmion states with arbitrary topological charges. Taking into account the hierarchy of effective spin interactions, an analytical theory on the optimal sizes of such states -- the higher-order magnetic skyrmions -- is developed for axially symmetric magnetic fields of the form $h(r) \sim r^{\beta}$ with $\beta \in \mathbb{R}$.Comment: 20 pages, 10 figures, 75 reference

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

Near-Infrared Fluorescent Proteins and Their Applications

High transparency, low light-scattering, and low autofluorescence of mammalian tissues in the near-infrared (NIR) spectral range (650-900 nm) open a possibility for in vivo imaging of biological processes at the micro-and macroscales to address basic and applied problems in biology and biomedicine. Recently, probes that absorb and fluoresce in the NIR optical range have been engineered using bacterial phytochromes-natural NIR light-absorbing photoreceptors that regulate metabolism in bacteria. Since the chromophore in all these proteins is biliverdin, a natural product of heme catabolism in mammalian cells, they can be used as genetically encoded fluorescent probes, similarly to GFP-like fluorescent proteins. In this review, we discuss photophysical and biochemical properties of NIR fluorescent proteins, reporters, and biosensors and analyze their characteristics required for expression of these molecules in mammalian cells. Structural features and molecular engineering of NIR fluorescent probes are discussed. Applications of NIR fluorescent proteins and biosensors for studies of molecular processes in cells, as well as for tissue and organ visualization in whole-body imaging in vivo, are described. We specifically focus on the use of NIR fluorescent probes in advanced imaging technologies that combine fluorescence and bioluminescence methods with photoacoustic tomography.Peer reviewe

Near-Infrared Fluorescent Proteins : Multiplexing and Optogenetics across Scales

Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescentproteins(FPs) engineeredfrombacterialphytochromeshave become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.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

Оцінка прогностичних критеріїв перебігу псоріазу для подальшої корекції лікування пацієнтів у післяопераційному періоді

Мета. Дослідити імуногістохімічні зміни в шкірі хворих із псоріазом для прогнозування можливого загострення шкір- ного псоріатичного процесу після виконання оперативного втручання. Матеріали і методи. Проведено імуногістохімічне дослідження біопсійного матеріалу, взятого з ділянок шкірної псо- ріатичної висипки та інтактної шкіри у хворих із псоріазом до виконання оперативного втручання. Крім того, для по- рівняння результатів імуногістохімічних досліджень вивчено біопсійний матеріал шкіри передньої черевної стінки, взятий у 5 практично здорових осіб відповідного віку після герніопластики. Для оцінки характеру та поширеності міс- цевих клітинних імунних і запальних реакцій у шкірі хворих із псоріазом застосовували імуногістохімічні методики з визначенням експресії маркерів імунного запалення. Результати. Представлені результати дослідження змін імуногістохімічної експресії прозапальних біологічних мар- керів у шкірі хворих із псоріазом до виконання оперативного втручання з подальшим аналізом впливу такого стресо- вого фактора, як оперативне втручання, на перебіг шкірного псоріатичного процесу. Висновки. Доведено, що одним із механізмів загострення запальних реакцій у хворих із псоріазом є активація імуно- компетентних клітин, зокрема через Toll–рецептори, що дає можливість скоригувати тактику лікування пацієнтів у піс- ляопераційному періоді

Genesis of the Russia’s economy innovative development

The innovative development of economy in Russia is possible only if the way of development will be based on the objective basic components, i.e. the corresponding system of premises and factors determining the innovative process. Namely the latter ensure a transformation of the fundamental processes into the application ones, as well as the dynamic development of the economy under the new trends and patterns influenceyesBelgorod State Universit