The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing ...structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.
The maintenance of genome stability is crucial for cell homeostasis and tissue integrity. Numerous human neuropathologies display chronic inflammation in the central nervous system, set against a ...backdrop of genome instability, implying a close interplay between the DNA damage and immune responses in the context of neurological disease. Dissecting the molecular mechanisms of this crosstalk is essential for holistic understanding of neuroinflammatory pathways in genome instability disorders. Non-neuronal cell types, specifically microglia, are major drivers of neuroinflammation in the central nervous system with neuro-protective and -toxic capabilities. Here, we discuss how persistent DNA damage affects microglial homeostasis, zooming in on the cytosolic DNA sensing cGAS-STING pathway and the downstream inflammatory response, which can drive neurotoxic outcomes in the context of genome instability.
•Cytosolic DNA sensing cGAS-STING pathway is at the intersection between genome instability and immune signalling.•Microglia are the main source of cGAS-STING signalling in the central nervous system.•Replication- and transcription-associated DNA damage can drive microglial inflammation with neurotoxic consequences.•Targeting neuroinflammation downstream of STING may be beneficial in a variety of neurodegenerative disorders driven by genome instability.
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-
m
-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.
Protein-fragment complementation assays are used ubiquitously for probing protein-protein interactions. Most commonly, the reporter protein is split in two parts, which are then fused to the proteins ...of interest and can reassemble and provide a readout if the proteins of interest interact with each other. The currently known split fluorescent proteins either can be used only in aerobic conditions and assemble irreversibly, or require addition of exogenous chromophores, which complicates the design of experiments. In recent years, light-oxygen-voltage (LOV) domains of several photoreceptor proteins have been developed into flavin-based fluorescent proteins (FbFPs) that, under some circumstances, can outperform commonly used fluorescent proteins such as GFP. Here, we show that CagFbFP, a small thermostable FbFP based on a LOV domain-containing protein from
, can serve as a split fluorescent reporter. We use the available genetic and structural information to identify three loops between the conserved secondary structure elements, Aβ-Bβ, Eα-Fα, and Hβ-Iβ, that tolerate insertion of flexible poly-Gly/Ser segments and eventually splitting. We demonstrate that the designed split pairs, when fused to interacting proteins, are fluorescent
in
and human cells and have low background fluorescence. Our results enable probing protein-protein interactions in anaerobic conditions without using exogenous fluorophores and provide a basis for further development of LOV and PAS (Per-Arnt-Sim) domain-based fluorescent reporters and optogenetic tools.
This is a review of relevant Raman spectroscopy (RS) techniques and their use in structural biology, biophysics, cells, and tissues imaging towards development of various medical diagnostic tools, ...drug design, and other medical applications. Classical and contemporary structural studies of different water-soluble and membrane proteins, DNA, RNA, and their interactions and behavior in different systems were analyzed in terms of applicability of RS techniques and their complementarity to other corresponding methods. We show that RS is a powerful method that links the fundamental structural biology and its medical applications in cancer, cardiovascular, neurodegenerative, atherosclerotic, and other diseases. In particular, the key roles of RS in modern technologies of structure-based drug design are the detection and imaging of membrane protein microcrystals with the help of coherent anti-Stokes Raman scattering (CARS), which would help to further the development of protein structural crystallography and would result in a number of novel high-resolution structures of membrane proteins—drug targets; and, structural studies of photoactive membrane proteins (rhodopsins, photoreceptors, etc.) for the development of new optogenetic tools. Physical background and biomedical applications of spontaneous, stimulated, resonant, and surface- and tip-enhanced RS are also discussed. All of these techniques have been extensively developed during recent several decades. A number of interesting applications of CARS, resonant, and surface-enhanced Raman spectroscopy methods are also discussed.
Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their ...final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.
The light-driven sodium pump KR2, found in 2013 in the marine bacteria Krokinobacter eikastus, serves as a model protein for the studies of the sodium-pumping microbial rhodopsins (NaRs). KR2 ...possesses a unique NDQ (N112, D116, and Q123) set of the amino acid residues in the functionally relevant positions, named the NDQ motif. The N112 was shown to determine the Na+/H+ selectivity and pumping efficiency of the protein. Thus, N112A mutation converts KR2 into an outward proton pump. However, no structural data on the functional conversions of the light-driven sodium pumps are available at the moment. Here we present the crystal structure of the N112A mutant of KR2 in the ground state at the resolution of 2.4 Å. The structure revealed a minor deflection in the central part of the helix C and a double conformation of the L74 residue in the mutant. The organization of the retinal Schiff base and neighboring water molecules is preserved in the ground state of KR2-N112A. The presented data provide structural insights into the effects of the alterations of the characteristic NDQ motif of NaRs. Our findings also demonstrate that for the rational design of the KR2 variants with modified ion selectivity for optogenetic applications, the structures of the intermediate states of both the protein and its functional variants are required.
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