ATP synthases are unique rotatory molecular machines that supply biochemical reactions with adenosine triphosphate (ATP)—the universal “currency”, which cells use for synthesis of vital molecules and ...sustaining life. ATP synthases of F-type (F
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) are found embedded in bacterial cellular membrane, in thylakoid membranes of chloroplasts, and in mitochondrial inner membranes in eukaryotes. The main functions of ATP synthases are control of the ATP synthesis and transmembrane potential. Although the key subunits of the enzyme remain highly conserved, subunit composition and structural organization of ATP synthases and their assemblies are significantly different. In addition, there are hypotheses that the enzyme might be involved in the formation of the mitochondrial permeability transition pore and play a role in regulation of the cell death processes. Dysfunctions of this enzyme lead to numerous severe disorders with high fatality levels. In our review, we focus on F
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-structure-based approach towards development of new therapies by using F
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structural features inherited by the representatives of this enzyme family from different taxonomy groups. We analyzed and systematized the most relevant information about the structural organization of F
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to discuss how this approach might help in the development of new therapies targeting ATP synthases and design tools for cellular bioenergetics control.
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by ...shifting them towards longer wavelengths. However, Ca
-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca
-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca
-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the "dark" pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v.
The studies of the functional properties of retinal-containing proteins often include experiments in model membrane systems, e.g., measurements of electric current through planar bilayer lipid ...membranes (BLMs) with proteoliposomes adsorbed on one of the membrane surfaces. However, the possibilities of this method have not been fully explored yet. We demonstrated that the voltage dependence of stationary photocurrents for two light-sensitive proteins, bacteriorhodopsin (bR) and channelrhodopsin 2 (ChR2), in the presence of protonophore had very different characteristics. In the case of the bR (proton pump), the photocurrent through the BLM did not change direction when the polarity of the applied voltage was switched. In the case of the photosensitive channel protein ChR2, the photocurrent increased with the increase in voltage and the current polarity changed with the change in the voltage polarity. The protonophore 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole (TTFB) was more efficient in the maximizing stationary photocurrents. In the presence of carbonyl cyanide-
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-chlorophenylhydrazone (CCCP), the amplitude of the measured photocurrents for bR significantly decreased, while in the case of ChR2, the photocurrents virtually disappeared. The difference between the effects of TTFB and CCCP was apparently due to the fact that, in contrast to TTFB, CCCP transfers protons across the liposome membranes with a higher rate than through the decane-containing BLM used as a surface for the proteoliposome adsorption.
People are constantly exposed to airborne fungal spores, including
conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, ...immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense
conidia and maintain homeostasis. The mechanisms by which IE-DCs contribute to regulating the antifungal immune response and controlling conidia dissemination are not understood. To clarify the role of IE-DCs in the balance between pathogen sensing and immune tolerance we investigated the
conidia distribution in optically cleared mouse lungs and estimated the kinetics of the local phagocytic response during the course of inflammation. MHCII
antigen-presenting cells, including IE-DCs, and CD11b
phagocytes were identified by immunohistochemistry and three-dimensional fluorescence confocal laser-scanning microscopy of conducting airway whole-mounts. Application of
conidia increased the number of CD11b
phagocytes in the conducting airway mucosa and induced the trafficking of these cells through the conducting airway wall to the luminal side of the epithelium. Some CD11b
phagocytes internalized conidia in the conducting airway lumen. During the migration through the airway wall, CD11b
phagocytes formed clusters. Permanently located in the airway wall IE-DCs contacted both single CD11b
phagocytes and clusters. Based on the spatiotemporal characteristics of the interactions between IE-DCs and CD11b
phagocytes, we provide a novel anatomical rationale for the contribution of IE-DCs to controlling the excessive phagocyte-mediated immune response rather than participating in pathogen uptake.
Membrane proteins (MPs) play vital roles in the function of cells and are also major drug targets. Structural information on proteins is vital for understanding their mechanism of function and is ...critical for the development of drugs. However, obtaining high‐resolution structures of membrane proteins, in particular, under native conditions is still a great challenge. In such cases, the low‐resolution methods small‐angle X‐ray and neutron scattering (SAXS and SANS) might provide valuable structural information. However, in some cases small‐angle scattering (SAS) provides ambiguous ab initio structural information if complementary measurements are not performed and/or a priori information on the protein is not taken into account. Understanding the nature of the limitations may help to overcome these problems. One of the main problems of SAS data analysis of solubilized membrane proteins is the contribution of the detergent belt surrounding the MP. Here, a comprehensive analysis of how the detergent belt contributes to the SAS data of a membrane‐protein complex of sensory rhodopsin II with its cognate transducer from Natronomonas pharaonis (NpSRII–NpHtrII) was performed. The influence of the polydispersity of NpSRII–NpHtrII oligomerization is the second problem that is addressed here. It is shown that inhomogeneity in the scattering length density of the detergent belt surrounding a membrane part of the complex and oligomerization polydispersity significantly impacts on SAXS and SANS profiles, and therefore on 3D ab initio structures. It is described how both problems can be taken into account to improve the quality of SAS data treatment. Since SAS data for MPs are usually obtained from solubilized proteins, and their detergent belt and, to a certain extent, oligomerization polydispersity are sufficiently common phenomena, the approaches proposed in this work might be used in SAS studies of different MPs.
Ambiguities in and the completeness of SAS data analysis of membrane proteins (MPs) are considered here in the case of the sensory rhodopsin II–transducer complex solubilized in a detergent. The contribution of the detergent belt surrounding MPs is one of the main problems in SAS data analysis of MPs; the second problem addressed here is the influence of oligomerization polydispersity (which is a sufficiently common phenomenon for MPs) on the quality of SAS structural analysis.
Microbial rhodopsin (MRs) ion channels and pumps have become invaluable optogenetic tools for neuroscience as well as biomedical applications. Recently, MR-optogenetics expanded towards subcellular ...organelles opening principally new opportunities in optogenetic control of intracellular metabolism and signaling
precise manipulations of organelle ion gradients using light. This new optogenetic field expands the opportunities for basic and medical studies of cancer, cardiovascular, and metabolic disorders, providing more detailed and accurate control of cell physiology. This review summarizes recent advances in studies of the cellular metabolic processes and signaling mediated by optogenetic tools targeting mitochondria, endoplasmic reticulum (ER), lysosomes, and synaptic vesicles. Finally, we discuss perspectives of such an optogenetic approach in both fundamental and applied research.
Two-component systems (TCS) are widespread signaling systems present in all domains of life. TCS typically consist of a signal receptor/transducer and a response regulator. The receptors (histidine ...kinases, chemoreceptors and photoreceptors) are often embedded in the membrane and have a similar modular structure. Chemoreceptors were shown to function in highly ordered arrays, with trimers of dimers being the smallest functional unit. However, much less is known about photoreceptors. Here, we use small-angle scattering (SAS) to show that detergent-solubilized sensory rhodopsin II in complex with its cognate transducer forms dimers at low salt concentration, which associate into trimers of dimers at higher buffer molarities. We then fit an atomistic model of the whole complex into the SAS data. The obtained results suggest that the trimer of dimers is "tripod"-shaped and that the contacts between the dimers occur only through their cytoplasmic regions, whereas the transmembrane regions remain unconnected.
Abstract
Despite remarkable progress, mainly due to the development of LCP and ‘bicelle’ crystallization, lack of structural information remains a bottleneck in membrane protein (MP) research. A ...major reason is the absence of complete understanding of the mechanism of crystallization. Here we present small-angle scattering studies of the evolution of the “bicelle” crystallization matrix in the course of MP crystal growth. Initially, the matrix corresponds to liquid-like bicelle state. However, after adding the precipitant, the crystallization matrix transforms to jelly-like state. The data suggest that this final phase is composed of interconnected ribbon-like bilayers, where crystals grow. A small amount of multilamellar phase appears, and its volume increases concomitantly with the volume of growing crystals. We suggest that the lamellar phase surrounds the crystals and is critical for crystal growth, which is also common for LCP crystallization. The study discloses mechanisms of “bicelle” MP crystallization and will support rational design of crystallization.
Bacteriorhodopsin (bR) provides light-driven vectorial proton transport across a cell membrane. Creation of electrochemical potential at the membrane is a universal step in energy transformation in a ...cell. Published atomic crystallographic models of early intermediate states of bR show a significant difference between them, and conclusions about pumping mechanisms have been contradictory. Here, we present a quantitative high-resolution crystallographic study of conformational changes in bR induced by X-ray absorption. It is shown that X-ray doses that are usually accumulated during data collection for intermediate-state studies are sufficient to significantly alter the structure of the protein. X-ray-induced changes occur primarily in the active site of bR. Structural modeling showed that X-ray absorption triggers retinal isomerization accompanied by the disappearance of electron densities corresponding to the water molecule W402 bound to the Schiff base. It is demonstrated that these and other X-ray-induced changes may mimic functional conformational changes of bR leading to misinterpretation of the earlier obtained X-ray crystallographic structures of photointermediates.
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Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-β peptide (Aβ) oligomers play a crucial role in AD pathogenesis. All
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Enantiomeric ...peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aβ aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aβ monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aβ sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aβ conversion into β-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.
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