A subgroup of light-driven sodium pumps with an additional Schiff base counterion.

Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.

Structural insights into light-driven anion pumping in cyanobacteria.

Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl-, I-, NO3-. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics.

Na+-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) harness [Na+], [K+], and [H+] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na+ ions, [Na+] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na+:substrate coupling in the EAAT homolog GltPh A lipidic cubic phase x-ray crystal structure of wild-type, Na+-only bound GltPh at 2.5-Å resolution revealed the fully open, outward-facing state primed for subsequent substrate binding. Simulations and kinetic experiments established that only the binding of two Na+ ions to the Na1 and Na3 sites ensures complete HP2 gate opening via a conformational selection-like mechanism and enables high-affinity substrate binding via electrostatic attraction. The combination of Na+-stabilized gate opening and electrostatic coupling of aspartate to Na+ binding provides a constant Na+:substrate transport stoichiometry over a broad range of neurotransmitter concentrations.

High-resolution structural insights into the heliorhodopsin family.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), has recently been discovered. Unlike in the known rhodopsins, in HeRs the N termini face the cytoplasm. The function of HeRs remains unknown. We present the structures of the bacterial HeR-48C12 in two states at the resolution of 1.5 Å, which highlight its remarkable difference from all known rhodopsins. The interior of HeR's extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (Schiff base cavity [SBC]) surrounded by charged amino acids and containing a cluster of water molecules, presumably being a primary proton acceptor from the Schiff base. At acidic pH, a planar triangular molecule (acetate) is present in the SBC. Structure-based bioinformatic analysis identified 10 subfamilies of HeRs, suggesting their diverse biological functions. The structures and available data suggest an enzymatic activity of HeR-48C12 subfamily and their possible involvement in fundamental redox biological processes.

New Insights on Signal Propagation by Sensory Rhodopsin II/Transducer Complex.

The complex of two membrane proteins, sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII), mediates negative phototaxis in halobacteria N. pharaonis. Upon light activation NpSRII triggers a signal transduction chain homologous to the two-component system in eubacterial chemotaxis. Here we report on crystal structures of the ground and active M-state of the complex in the space group I212121. We demonstrate that the relative orientation of symmetrical parts of the dimer is parallel ("U"-shaped) contrary to the gusset-like ("V"-shaped) form of the previously reported structures of the NpSRII/NpHtrII complex in the space group P21212, although the structures of the monomers taken individually are nearly the same. Computer modeling of the HAMP domain in the obtained "V"- and "U"-shaped structures revealed that only the "U"-shaped conformation allows for tight interactions of the receptor with the HAMP domain. This is in line with existing data and supports biological relevance of the "U" shape in the ground state. We suggest that the "V"-shaped structure may correspond to the active state of the complex and transition from the "U" to the "V"-shape of the receptor-transducer complex can be involved in signal transduction from the receptor to the signaling domain of NpHtrII.

High-resolution structure of a membrane protein transferred from amphipol to a lipidic mesophase.

Amphipols (APols) have become important tools for the stabilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs solubilized in APols would be of high importance for structural biology. However, despite considerable efforts, it is still not clear whether MP/APol complexes can form well-ordered crystals suitable for X-ray crystallography. In the present work, we show that an APol-trapped MP can be crystallized in meso. Bacteriorhodopsin (BR) trapped by APol A8-35 was mixed with a lipidic mesophase, and crystallization was induced by adding a precipitant. The crystals diffract beyond 2 Å. The structure of BR was solved to 2 Å and found to be indistinguishable from previous structures obtained after transfer from detergent solutions. We suggest the proposed protocol of in meso crystallization to be generally applicable to APol-trapped MPs.

Nanoparticle surface-enhanced Raman scattering of bacteriorhodopsin stabilized by amphipol A8-35.

Surface-enhanced Raman spectroscopy (SERS) has developed dramatically since its discovery in the 1970s, because of its power as an analytical tool for selective sensing of molecules adsorbed onto noble metal nanoparticles (NPs) and nanostructures, including at the single-molecule (SM) level. Despite the high importance of membrane proteins (MPs), SERS application to MPs has not really been studied, due to the great handling difficulties resulting from the amphiphilic nature of MPs. The ability of amphipols (APols) to trap MPs and keep them soluble, stable, and functional opens up onto highly interesting applications for SERS studies, possibly at the SM level. This seems to be feasible since single APol-trapped MPs can fit into gaps between noble metal NPs, or in other gap-containing SERS substrates, whereby the enhancement of Raman scattering signal may be sufficient for SM sensitivity. The goal of the present study is to give a proof of concept of SERS with APol-stabilized MPs, using bacteriorhodopsin (BR) as a model. BR trapped by APol A8-35 remains functional even after partial drying at a low humidity. A dried mixture of silver Lee-Meisel colloid NPs and BR/A8-35 complexes give rise to SERS with an average enhancement factor in excess of 10(2). SERS spectra resemble non-SERS spectra of a dried sample of BR/APol complexes.

Ground state structure of D75N mutant of sensory rhodopsin II in complex with its cognate transducer.

The complex of sensory rhodopsin II (NpSRII) with its cognate transducer (NpHtrII) mediates negative phototaxis in halobacteria Natronomonas pharaonis. Upon light activation NpSRII triggers, by means of NpHtrII, a signal transduction chain homologous to the two component system in eubacterial chemotaxis. Here we report on the crystal structure of the ground state of the mutant NpSRII-D75N/NpHtrII complex in the space group I212121. Mutations of this aspartic acid in light-driven proton pumps dramatically modify or/and inhibit protein functions. However, in vivo studies show that the similar D75N mutation retains functionality of the NpSRII/NpHtrII complex. The structure provides the molecular basis for the explanation of the unexpected observation that the wild and the mutant complexes display identical physiological response on light excitation.

[Immunocytochemical visualization of P185(HER2) receptor by antibody fused with dibarnase and conjugate of barstar with colloidal gold].

We studied the localization of transmembrane receptor P185(HER2) in SKOV-3 and BT-474 cancer cells by fluorescence, confocal and electron immunomicroscopy. P185(HER2) is a marker of breast and ovarian tumors, it is considered as a target for anticancer therapy. It is extremely important to choose a universal immunicytotoxic agent applicable, first, to study the distribution of P185(HER2) in cancer cells, secondly, to remove P185(HER2) from the cell surface and, thirdly, to eliminate target cells. In this work for visualization of P185HER2 We prOposed immunocytotoxic system, consisting of the monoclonal miniantibody 4D5 scFv to extracellular P185E domain fused with two molecules of barnase (ribonuclease from Bacillus amyloliquefaciens) and of its specific inhibitor barstar. Fluorescence microscopy has showed that the module 4D5 scFv-dibarnase:barstar efficiently identified P185(HER2) on the surface of cancer cells. It was revealed by confocal microscopy that interaction with 4D5 scFv-dibarnase lead to internalization of P185(HER2). The localization of P185(HER) in human ovarian carcinoma cells SKOV-3 and breast carcinoma cells BT-474 was compared by electron microscopy using 4D5 scFv-dibarnase:barstar-Au and 4D5 scFv-dibarnase-Au complexes. P185(HER) distributed on the cell surface unequally with preferential localization on protrusions or close to their bases and in contacts between protrusions and cell membrane. At 37 degrees C, P185(HER2) internalized through coated pits and vesicles and concentrated in the endosomes and multivesicular bodies in the cells of both cell lines, as well as in lysosomes in cells BT-474.

Spin label method reveals barnase-barstar interaction: a temperature and viscosity dependence approach.

Spin labeling was used to study the protein-protein interaction between the enzyme barnase (Bn) and its inhibitor barstar (Bs). A mutant of barstar (C40A), which contains only one cysteine, C82, located near the Bn-Bs contact region, was selectively modified by two spin labels having different lengths and structures of the flexible tether. The formation of a strong protein complex resulted in significant restriction of spin label mobility at the C82 residue of barstar, as indicated by notable changes in the recorded EPR spectra. The dependence of the separation between broad outer peaks of the EPR spectra on viscosity at constant temperature was used to evaluate the order parameter S and the rotational correlation time tau (a temperature-viscosity dependence approach). The order parameter S, which characterizes fast reorientation of a spin label relative to the protein molecule, sharply increases and approaches unity when Bs binds to Bn. In addition, formation of a Bs-Bs complex was observed; it is also accompanied by restriction of spin label mobility. At the same time, the rotational correlation times tau of spin-labeled Bs, its complex with Bn, and the Bs dimer in solution agree well with their molecular masses. This indicates that barstar, its complex with barnase, and barstar dimer are rigid protein entities. The described approach is suitable for studying any spin-labeled macromolecular complexes.

Dynamic spin label study of the barstar-barnase complex.

The dynamic spin label method was used to study protein-protein interactions in the model complex of the enzyme barnase (Bn) with its inhibitor barstar. The C40A mutant of barstar (Bs) containing a single cysteine residue was modified with two different spin labels varying in length and structure of a flexible linker. Each spin label was selectively bound to the Cys82 residue, located near the Bn-Bs contact site. The formation of the stable protein complex between Bn and spin labeled Bs was accompanied by a substantial restriction of spin label mobility, indicated by remarkable changes in the registered EPR spectra. Order parameter, S, as an estimate of rapid reorientation of spin label relative to protein molecule, was sharply increasing approaching 1. However, the rotational correlation time tau for spin-labeled Bs and its complex with Bn in solution corresponded precisely to their molecular weights. These data indicate that both Bs and its complex with Bn are rigid protein entities. Spin labels attached to Bs in close proximity to an interface of interaction with Bn, regardless of its structure, undergo significant restriction of mobility by the environment of the contact site of the two proteins. The results show that this approach can be used to investigate fusion proteins containing Bn or Bs.

[Antiviral activity of Bacillus intermedius RNAase in guinea-pigs and rabbits infected with outdoor rabies virus].

Single injection of Bacillus intermedius RNAase in a dose of 5 mg/kg could protect 40 and 50-70% of the outdoor rabies virus-preinfected guinea-pigs and rabbits, respectively. In the control group there were 100 and 75-100% deaths of the RNAase-untreated guinea-pigs and rabbits, respectively. Animal protection was observed only when RNAase was injected into the site of viral administration. The intramuscular injection of RNAase, other than the site of viral administration failed to protect the infected animals. The efficacy (75%) of RNAase injected into the rabbits was similar 1 and 24 hours after animal infection.

[The antiviral activity of RNAse Bacillus intermedius in experiments with mice preinfected with street rabies virus]. / Protivovirusnaia aktivnost' RNKazy Bacillus intermedius v éksperimentakh na myshakh linii CBA, predvaritel'no zarazhennykh ulichnym virusom beshenstva.

RNAse Bacillus intermedius, when administered once and according to 11 repeated experiments, protected the preliminarily infected CBA mice with street rabies virus (protection of 40-67%; p < 0.01-0.001). A reliable protection of Animals was registered only when RNAse was administered intramuscularly at the virus introduction spot; it was not effective, when the bacterial RNAse was injected in the brain, vein, under the skin or in muscles of a non-infected extremity. Neither did it produce any suppressive effect on the vaccinal antirabic immunity.

Silencing of a beta-1,3-glucanase transgene is overcome during seed formation.

Expression of a beta-1,3-glucanase transgene (gn1) driven by the CaMV 35S promoter is silenced in the T17 homozygous tobacco transgenic line. This silencing process is post-transcriptionally regulated and subject to developmental control. We have examined this phenomenon to investigate the developmental pathways involved in suppression and reactivation of gn1 expression as well as to identify the plant tissues where these processes occur. Analysis of beta-1,3-glucanase activity and gene expression have allowed us to determine that suppression of gn1 is a very efficient process reducing the steady-state gn1 mRNA level, simultaneously, in all leaves of the plant. Gene silencing occurs a few weeks after seed germination, and is maintained throughout vegetative growth and floral development. Expression of gn1 is restored in the maturing fruit some time after fertilization. In situ hybridization analyses show that expression of gn1 is restored within the developing seeds in tissues derived from meiotically divided cells. In contrast to the high level of expression found in seedlings obtained from germinated T17 homozygous seeds, the expression of gn1 is not reactivated in plantlets regenerated in vitro from leaf explants of suppressed T17 homozygous plants that is, in plant tissues obtained by mitotic division. Thus, reactivation of gn1 expression specifically occurs along the developmental programme controlling sexual reproduction and likely throughout epigenetic modifications affecting the state of gene expression during meiosis.

Structure and induction pattern of a novel proteinase inhibitor class II gene of tobacco.

A cDNA and a corresponding genomic clone encoding a protein with partial identity to type II proteinase inhibitors from potato, tomato and Nicotiana alata, were isolated from tobacco libraries. The protein of 197 amino acids contains a putative signal peptide of 24 residues and three homologous domains, each with a different reactive site. The tobacco PI-II gene is not expressed in leaves of healthy plants, but is locally induced in leaves subjected to different types of stress (TMV infection, wounding, UV irradiation) and upon ethephon treatment. As opposed to the analogous PI-II genes of potato and tomato, the tobacco gene is not systemically induced by wounding or pathogenic infection. A far-upstream region in the PI-II promoter, containing various direct and indirect repeats, shares considerable sequence similarity to a similar region in the stress-inducible Cu/Zn-superoxide dismutase gene of N. plumbaginifolia.

Characterization of the Reversible Inactivation of Ankistrodesmus braunii Nitrate Reductase by Hydroxylamine.

The photoreversible nature of the regulation of nitrate reductase is one of the most interesting features of this enzyme. As well as other chemicals, NH(2)OH reversibly inactivates the reduced form of nitrate reductase from Ankistrodesmus braunii. From the partial activities of the enzyme, only terminal nitrate reductase is affected by NH(2)OH. To demonstrate that the terminal activity was readily inactivted by NH(2)OH, the necessary reductants of the terminal part of the enzyme had to be cleared of dithionite since this compound reacts chemically with NH(2)OH. Photoreduced flavins and electrochemically reduced methyl viologen sustain very effective inactivation of terminal nitrate reductase activity, even if the enzyme was previously deprived of its NADH-dehydrogenase activity. The early inhibition of nitrate reductase by NH(2)OH appears to be competitive versus NO(3) (-). Since NO(3) (-), as well as cyanate, carbamyl phosphate and azide (competitive inhibitors of nitrate reductase versus NO(3) (-)), protect the enzyme from NH(2)OH inactivation, it is suggested that NH(2)OH binds to the nitrate active site. The NH(2)OH-inactivated enzyme was photoreactivated in the presence of flavins, although slower than when the enzyme was previously inactivated with CN(-). NH(2)OH and NADH concentrations required for full inactivation of nitrate reductase appear to be low enough to potentially consider this inactivation process of physiological significance.