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.
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.
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.
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to ...produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs - a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia.
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.
Phytoplankton is the base of the marine food chain as well as oxygen and carbon cycles and thus plays a global role in climate and ecology. Nucleocytoplasmic Large DNA Viruses that infect ...phytoplankton organisms and regulate the phytoplankton dynamics encompass genes of rhodopsins of two distinct families. Here, we present a functional and structural characterization of two proteins of viral rhodopsin group 1, OLPVR1 and VirChR1. Functional analysis of VirChR1 shows that it is a highly selective, Na
/K
-conducting channel and, in contrast to known cation channelrhodopsins, it is impermeable to Ca
ions. We show that, upon illumination, VirChR1 is able to drive neural firing. The 1.4 Å resolution structure of OLPVR1 reveals remarkable differences from the known channelrhodopsins and a unique ion-conducting pathway. Thus, viral rhodopsins 1 represent a unique, large group of light-gated channels (viral channelrhodopsins, VirChR1s). In nature, VirChR1s likely mediate phototaxis of algae enhancing the host anabolic processes to support virus reproduction, and therefore, might play a major role in global phytoplankton dynamics. Moreover, VirChR1s have unique potential for optogenetics as they lack possibly noxious Ca
permeability.
This paper is referenced to a design of a gyrocardiography unit and methods of a sternum gyrocardiogramm signal measurement and processing. The analysis of an information capacity of the ...gyrocardiogram is carried out in comparison with elecrocardiography and seismocardiography signals. It is provided that the gyrocardiorgamm has properties of signals and so can be used for information analysis for noninvasive diagnostics of diseases of internal organs.
The research studies of e-beam sustained discharge gas lasers initiated by Nikolai Basov in early 1970s made it possible to develop lasers based on the fundamental transitions of carbon monoxide ...molecule with high laser power and efficiency. Soon, under his scientific supervision, lasing on first-overtone transitions of CO molecule was obtained. Following these results, about ten years ago a compact slab CO laser excited by a repetitively pulsed capacitive RF discharge and cryogenic cooling of its electrodes, operating without forced pumping of its active medium was for the first time developed at the N.G. Basov Quantum Radiophysics Division of the P.N. Lebedev Physical Institute. At present, the average power of such lasers with active medium volume of ~ 35 cm
3
reaches 40 W on CO molecule fundamental transitions (in the wavelength range of 5.06–5.92 μm) and 6 W on first-overtone transitions (λ = 2.60–3.05 μm). Under Q-switching, those repetitively pulsed lasers allow one to obtain laser emission with a peak power up to 5 kW which made it possible to apply them in experiments on laser radiation frequency conversion in nonlinear crystals into the spectral range of ~ 2–20 μm.
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