Phytochromes are biological red/far-red light sensors found in many organisms. Photoisomerization of the linear methine-bridged tetrapyrrole triggers transient proton translocation events in the ...chromophore binding pocket (CBP) leading to major conformational changes of the protein matrix that are in turn associated with signaling. By combining pH-dependent resonance Raman and UV–visible absorption spectroscopy, we analyzed protonation-dependent equilibria in the CBP of Cph1 involving the proposed Pr-I and Pr-II substates that prevail below and above pH 7.5, respectively. The protonation pattern and vibrational properties of these states were further characterized by means of hybrid quantum mechanics/molecular mechanics calculations. From this combined experimental–theoretical study, we were able to identify His260 as the key residue controlling pH-dependent equilibria. This residue is not only responsible for the conformational heterogeneity of CBP in the Pr state of prokaryotic phytochromes, discussed extensively in the past, but it constitutes the sink and source of protons in the proton release/uptake mechanism involving the tetrapyrrole chromophore which finally leads to the formation of the Pfr state. Thus, this work provides valuable information that may guide further experiments toward the understanding of the specific role of protons in controlling structure and function of phytochromes in general.
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IJS, KILJ, NUK, PNG, UL, UM
Cyanobacteriochromes are members of the phytochrome superfamily. In contrast to classical phytochromes, these small photosensors display a considerable variability of electronic absorption maxima. We ...have studied the light-induced conversions of the second GAF domain of AnPixJ, AnPixJg2, a phycocyanobilin-binding protein from the cyanobacterium Anabaena PCC 7120, using low-temperature resonance Raman spectroscopy combined with molecular dynamics simulations. AnPixJg2 is formed biosynthetically as a red-absorbing form (Pr) and can be photoconverted into a green-absorbing form (Pg). Forward and backward phototransformations involve the same reaction sequences and intermediates of similar cofactor structures as the corresponding processes in canonical phytochromes, including a transient cofactor deprotonation. Whereas the cofactor of the Pr state shows far-reaching similarities to the Pr states of classical phytochromes, the Pg form displays significant upshifts of the methine bridge stretching frequencies concomitant to the hypsochromically shifted absorption maximum. However, the cofactor in Pg is protonated and adopts a conformation very similar to the Pfr state of classical phytochromes. The spectral differences are probably related to an increased solvent accessibility of the chromophore which may reduce the π-electron delocalization in the phycocyanobilin and thus raise the energies of the first electronic transition and the methine bridge stretching modes. Molecular dynamics simulations suggest that the Z → E photoisomerization of the chromophore at the C–D methine bridge alters the interactions with the nearby Trp90 which in turn may act as a gate, allowing the influx of water molecules into the chromophore pocket. Such a mechanism of color tuning AnPixJg2 is unique among the cyanobacteriochromes studied so far.
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Insights in active sites: Hydrogen‐conversion by hydrogenase is mediated by a sophisticated, metal‐containing catalytic center. Resonance Raman spectroscopy is used for the first time in the ...characterization of the active site of these biocatalysts. An integrated spectroscopic and computational approach gives insights into structural and photochemical properties of the active site of an oxygen‐tolerant NiFe hydrogenase.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Phytochromes are modular photoreceptors of plants, bacteria and fungi that use light as a source of information to regulate fundamental physiological processes. Interconversion between the active and ...inactive states is accomplished by a photoinduced reaction sequence which couples the sensor with the output module. However, the underlying molecular mechanism is yet not fully understood due to the lack of structural data of functionally relevant intermediate states. Here we report the crystal structure of a Meta-F intermediate state of an Agp2 variant from Agrobacterium fabrum. This intermediate, the identity of which was verified by resonance Raman spectroscopy, was formed by irradiation of the parent Pfr state and displays significant reorientations of almost all amino acids surrounding the chromophore. Structural comparisons allow identifying structural motifs that might serve as conformational switch for initiating the functional secondary structure change that is linked to the (de-)activation of these photoreceptors.
Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the ...enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from
Agrobacterium fabrum
. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield,
i.e.
the events of tongue refolding per absorbed photons, is lower by a factor of
ca.
two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.
Time-resolved vibrational spectroscopies reveal that phototransformation of the Pfr dark state of bacterial phytochrome Agp2 follows a branched mechanism with a productive and non-productive pathway.
Summary
T helper type 17 lymphocytes (Th17 cells) infiltrate the central nervous system (CNS), induce inflammation and demyelination and play a pivotal role in the pathogenesis of multiple sclerosis. ...Sialomucin CD43 is highly expressed in Th17 cells and mediates adhesion to endothelial selectin (E‐selectin), an initiating step in Th17 cell recruitment to sites of inflammation. CD43−/− mice have impaired Th17 cell recruitment to the CNS and are protected from experimental autoimmune encephalomyelitis (EAE), the mouse model of multiple sclerosis. However, E‐selectin is dispensable for the development of EAE, in contrast to intercellular and vascular cell adhesion molecules (ICAM‐1 and VCAM‐1). We report that CD43−/− mice have decreased demyelination and T‐cell infiltration, but similar up‐regulation of ICAM‐1 and VCAM‐1 in the spinal cord, compared with wild‐type (WT) mice, at the initiation of EAE. CD43−/− Th17 cells have impaired adhesion to ICAM‐1 under flow conditions in vitro, despite having similar expression of LFA‐1, the main T‐cell ligand for ICAM‐1, as WT Th17 cells. Regardless of the route of integrin activation, CD43−/− Th17 cell firm arrest on ICAM‐1 was comparable to that of WT Th17 cells, but CD43−/− Th17 cells failed to optimally apically migrate on immobilized ICAM‐1‐coated coverslips and endothelial cells, and to transmigrate under shear flow conditions in an ICAM‐1‐dependent manner. Collectively, these findings unveil novel roles for CD43, facilitating adhesion of Th17 cells to ICAM‐1 and modulating apical and transendothelial migration, as mechanisms potentially responsible for Th17 cell recruitment to sites of inflammation such as the CNS.
Our manuscript contributes to the field of Th17 cell recruitment during inflammation and documents a new role for sialomucin CD43 in facilitating Th17 cell adhesion, apical migration and transendothelial migration in an LFA‐1/ICAM‐1‐dependent manner.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Sialomucin CD43 is a transmembrane protein differentially expressed in leukocytes that include innate and adaptive immune cells. Among a variety of cellular processes, CD43 participates in T cell ...adhesion to vascular endothelial cells and contributes to the progression of experimental autoimmunity. Sequential infiltration of myeloid cells and T cells in the heart is a hallmark of cardiac inflammation and heart failure (HF). Here, we report that CD43-/- mice have improved survival to HF induced by transverse aortic constriction (TAC). This enhanced survival is associated with improved systolic function, decreased cardiac fibrosis, and significantly reduced T cell cardiac infiltration in response to TAC compared to control wild-type (WT) mice. Lack of CD43 did not alter the number of myeloid cells in the heart, but resulted in decreased cardiac CXCL10 expression, a chemoattractant for T cells, and in a monocyte shift to anti-inflammatory macrophages
. Collectively, these findings unveil a novel role for CD43 in adverse cardiac remodeling in pressure overload induced HF through modulation of cardiac T cell inflammation.
The second GAF domain of AnPixJ, AnPixJg2, a bilin-binding protein from the cyanobacterium Anabaena PCC 7120, undergoes a photoinduced interconversion between a red-absorbing state, Pr, and a ...green-absorbing state, Pg. Combining ultraviolet–vis (UV–vis), infrared, resonance Raman (RR), and magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, we have studied this cyanobacteriochrome (CBCR) assembled with phycocyanobilin (PCB) either in vivo or in vitro. In both assembly routes, the spectroscopic data of the Pr state reveal nearly identical chromophore structures with a protonated (cationic) bilin. However, unlike the native (in vivo assembly) Pg photoproduct, in which the bilin retains protonation, the Pg generated from the in vitro-assembled AnPixJg2 harbors a deprotonated (neutral) bilin chromophore at pH 7.8. IR difference spectroscopy further reveals the transfer of a proton from the bilin to a side-chain carboxylate on an amino acid, probably Asp291. Besides the change in protonation state, the bilin structure is very similar in the in vitro- and in vivo-assembled Pg photoproducts. The chromophore of the in vitro Pg becomes protonated when the pH is increased to 10, presumably because of a partial reversal of protein misfolding. Most remarkably, the electronic transitions remain unchanged and are very similar to those of the native Pg. Thus, bilin protonation is not a key parameter for controlling the energies of the electronic transitions in AnPixJg2. Possible alternative molecular mechanisms for color tuning are discussed.
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IJS, KILJ, NUK, PNG, UL, UM
Increasing pressure on water resources is driving the development of technology to improve water-use efficiency in irrigation. Uptake of these technological advances are essential to ensure long-term ...water security in catchments, particularly in water-scarce regions and where agricultural activities and urban centres compete for the same resources. Research suggests that uptake of technology lags far behind the development of new products. The study presented in this paper interviewed 29 commercial farmers from the water-scarce Central Breede River Valley in South Africa to investigate their reasons behind the use or non-use of irrigation technology for scheduling, and in particular the uptake of a free, government-funded remote-sensing service called FruitLook. Evaluating the uptake of a free service eliminates monetary cost as one key barrier to uptake. In-depth interviews revealed a high uptake of technology (83%), but use of only one type – soil water measurement. Among the farmers that use water-use efficiency technology, 78% use the same probe service provider. Perceived accuracy and ease of use, as well as personalised after-sales service are the key reasons for this probe’s popularity. While 86% of the farmers have heard about FruitLook, only one farmer uses it for irrigation purposes. The non-use of the free service can mainly be attributed to the time cost associated with the product’s initial set-up, use, and interpretation of information. The study revealed that the integration of information from various products is essential for farmers – too much information in different formats is too time-costly. Developers of new technology should focus on these latter two findings to improve the likelihood of new product uptake.
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NUK, OILJ, SAZU, UKNU, UL, UM, UPUK
Frequently, the plants used for the management of contaminated soil are those native to the area of action and provide a range of ecosystem services (ES). However, in many cases the sustainability of ...the afforestation planning and the provision of ES are not evaluated under scenarios of climate change (CC). In the present research work, we propose a fast‐track methodology for evaluating the potential distribution of forest species and provisioning of carbon sequestration under soil contaminated by trace elements. Furthermore, the effect of CC on the potential distribution was evaluated through the calibration of the Sierra model from MicroLEIS‐DSS. Seven tree species were selected due to their tolerance of soil contaminated by trace elements during the afforestation planning using the vegetation cover maps (1998–2013) of the Guadiamar Green Corridor. Additionally, compilation, standardization, and cluster process of soil database were applied following INSPIRE, EU‐HYDI, and JRC recommendations. Finally, the carbon sequestration rate of vegetation cover has been calculated according to IPCC methodology. The results show the loss of forest aptitude (Eucalyptus, P. pinea, P. alba, Quercus) under scenario of CC and decreasing of the ES provided. The proposed methodology could support the decision making to determine forest species suited for the regulation of contaminated soils and the provision of ES at spatiotemporal scales.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
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