Mechanisms of inward transmembrane proton translocation.

Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge. It requires true-atomic-resolution structures of all key proton-conducting states. Here we present a comprehensive function-structure study of a light-driven bacterial inward proton pump, xenorhodopsin, from Bacillus coahuilensis in all major proton-conducting states. The structures reveal that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. We demonstrate the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. The results might also be of interest for optogenetics since xenorhodopsins are the only alternative tools to fire neurons.

Nanostructured TiAlCuN and TiAlCuCN coatings for spacecraft: effects of reactive magnetron deposition regimes and compositions.

Spacecraft are exposed to a number of factors in the outer space: irradiation by electron flows, high-energy ions, solar electromagnetic radiation, plasma irradiation, and a stream of meteorite particles. All these factors initiate various physical and chemical processes in spacecraft materials, which can eventually lead to failure. To ensure reliable operation of spacecraft, it is necessary to use protective coatings and special radiation-resistant materials. TiAlCuN and TiAlCuCN coatings were formed by reactive magnetron sputtering on different substrates: single-crystal silicon and Titanium Grade 2 wafers. Nitrogen was used as a reactive gas to form nitride coatings and acetylene was used to form carbonitride coatings. The elemental composition was studied by energy-dispersive X-ray (EDX) spectroscopy. The structural-phase state of the coatings was examined by X-ray diffraction (XRD) and scanning electron microscopy (SEM). Mechanical properties, such as hardness and Young modulus, were investigated by nanoindentation using a CSM Instruments Nanohardness Tester NHT2. The influence of deposition parameters, such as Ti and Al contents, the degree of reactivity α, and carbonitride formation on the structure and their mechanical properties were considered. It was detected that Cu addition to the coatings has effects on crystallite and growth column size refinement in comparison with the TiAlN and TiAlCN analogues due to its segregation along crystalline boundaries, and thus, imparts better mechanical characteristics. The hardness of TiAlCuN and TiAlCuCN coatings varies in the range of H = 25-36 GPa and Young modulus - E = 176-268 GPa. The impact strength index and the H/E* ratio, as well as the plastic deformation resistance index H3/E*2, were calculated. Due to their high mechanical properties, the formed nitride and carbonitride coatings are promising for use in space technologies.

Mirror proteorhodopsins.

Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name "mirror proteorhodopsins", from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest.

Time-Resolved UV-VIS Spectroscopy of Microbial Rhodopsins.

Absorption of light quanta by microbial rhodopsins (or more generally by retinal proteins) leads to conversion of the light energy to the generation of transmembrane anion or cation gradients, optically gated channels, or signal states in photoreception. All these processes are accompanied by series of reaction steps with half-times ranging from femtoseconds to seconds or longer (photocycles). The number of these steps and their kinetic and spectral properties are the essential experimental information required for determination of the mechanism of light energy conversion in these proteins. Here we describe experiments and data analysis providing this information.

Distinct actin-tropomyosin cofilament populations drive the functional diversification of cytoskeletal myosin motor complexes.

The effects of N-terminal acetylation of the high molecular weight tropomyosin isoforms Tpm1.6 and Tpm2.1 and the low molecular weight isoforms Tpm1.12, Tpm3.1, and Tpm4.2 on the actin affinity and the thermal stability of actin-tropomyosin cofilaments are described. Furthermore, we show how the exchange of cytoskeletal tropomyosin isoforms and their N-terminal acetylation affects the kinetic and chemomechanical properties of cytoskeletal actin-tropomyosin-myosin complexes. Our results reveal the extent to which the different actin-tropomyosin-myosin complexes differ in their kinetic and functional properties. The maximum sliding velocity of the actin filament as well as the optimal motor density for continuous unidirectional movement, parameters that were previously considered to be unique and invariant properties of each myosin isoform, are shown to be influenced by the exchange of the tropomyosin isoform and the N-terminal acetylation of tropomyosin.

True-atomic-resolution insights into the structure and functional role of linear chains and low-barrier hydrogen bonds in proteins.

Hydrogen bonds are fundamental to the structure and function of biological macromolecules and have been explored in detail. The chains of hydrogen bonds (CHBs) and low-barrier hydrogen bonds (LBHBs) were proposed to play essential roles in enzyme catalysis and proton transport. However, high-resolution structural data from CHBs and LBHBs is limited. The challenge is that their 'visualization' requires ultrahigh-resolution structures of the ground and functionally important intermediate states to identify proton translocation events and perform their structural assignment. Our true-atomic-resolution structures of the light-driven proton pump bacteriorhodopsin, a model in studies of proton transport, show that CHBs and LBHBs not only serve as proton pathways, but also are indispensable for long-range communications, signaling and proton storage in proteins. The complete picture of CHBs and LBHBs discloses their multifunctional roles in providing protein functions and presents a consistent picture of proton transport and storage resolving long-standing debates and controversies.

Structure-based insights into evolution of rhodopsins.

Rhodopsins, most of which are proton pumps generating transmembrane electrochemical proton gradients, span all three domains of life, are abundant in the biosphere, and could play a crucial role in the early evolution of life on earth. Whereas archaeal and bacterial proton pumps are among the best structurally characterized proteins, rhodopsins from unicellular eukaryotes have not been well characterized. To fill this gap in the current understanding of the proton pumps and to gain insight into the evolution of rhodopsins using a structure-based approach, we performed a structural and functional analysis of the light-driven proton pump LR (Mac) from the pathogenic fungus Leptosphaeria maculans. The first high-resolution structure of fungi rhodopsin and its functional properties reveal the striking similarity of its membrane part to archaeal but not to bacterial rhodopsins. We show that an unusually long N-terminal region stabilizes the protein through direct interaction with its extracellular loop (ECL2). We compare to our knowledge all available structures and sequences of outward light-driven proton pumps and show that eukaryotic and archaeal proton pumps, most likely, share a common ancestor.

Structural and Biochemical Characterization of a Dye-Decolorizing Peroxidase from Dictyostelium discoideum.

A novel cytoplasmic dye-decolorizing peroxidase from Dictyostelium discoideum was investigated that oxidizes anthraquinone dyes, lignin model compounds, and general peroxidase substrates such as ABTS efficiently. Unlike related enzymes, an aspartate residue replaces the first glycine of the conserved GXXDG motif in Dictyostelium DyPA. In solution, Dictyostelium DyPA exists as a stable dimer with the side chain of Asp146 contributing to the stabilization of the dimer interface by extending the hydrogen bond network connecting two monomers. To gain mechanistic insights, we solved the Dictyostelium DyPA structures in the absence of substrate as well as in the presence of potassium cyanide and veratryl alcohol to 1.7, 1.85, and 1.6 Å resolution, respectively. The active site of Dictyostelium DyPA has a hexa-coordinated heme iron with a histidine residue at the proximal axial position and either an activated oxygen or CN- molecule at the distal axial position. Asp149 is in an optimal conformation to accept a proton from H2O2 during the formation of compound I. Two potential distal solvent channels and a conserved shallow pocket leading to the heme molecule were found in Dictyostelium DyPA. Further, we identified two substrate-binding pockets per monomer in Dictyostelium DyPA at the dimer interface. Long-range electron transfer pathways associated with a hydrogen-bonding network that connects the substrate-binding sites with the heme moiety are described.

Viral rhodopsins 1 are an unique family of light-gated cation channels.

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 Ca2+ 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 Ca2+ permeability.

Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function.

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.

Unique structure and function of viral rhodopsins.

Recently, two groups of rhodopsin genes were identified in large double-stranded DNA viruses. The structure and function of viral rhodopsins are unknown. We present functional characterization and high-resolution structure of an Organic Lake Phycodnavirus rhodopsin II (OLPVRII) of group 2. It forms a pentamer, with a symmetrical, bottle-like central channel with the narrow vestibule in the cytoplasmic part covered by a ring of 5 arginines, whereas 5 phenylalanines form a hydrophobic barrier in its exit. The proton donor E42 is placed in the helix B. The structure is unique among the known rhodopsins. Structural and functional data and molecular dynamics suggest that OLPVRII might be a light-gated pentameric ion channel analogous to pentameric ligand-gated ion channels, however, future patch clamp experiments should prove this directly. The data shed light on a fundamentally distinct branch of rhodopsins and may contribute to the understanding of virus-host interactions in ecologically important marine protists.

Microbial Halorhodopsins: Light-Driven Chloride Pumps.

Early research on the four microbial rhodopsins discovered in the archaeal Halobacterium salinarum revealed a structural template that served as a scaffold for two different functions: light-driven ion transport and phototaxis. Bacteriorhodopsin and halorhodopsin are proton and chloride pumps, respectively, while sensory rhodopsin I and II are responsible for phototactic behavior of the archaea. Halorhodopsins have been identified in various other species. Besides this group of archaeal halorhodopsins distinct chloride transporting rhodopsins groups have recently been identified in other organism like Flavobacteria or Cyanobacteria. Halorhodopsin from Natronomonas pharaonis is the best-studied homologue because of its facile expression and purification and its advantageous properties, which was the reason to introduce this protein as neural silencer into the new field of optogenetics. Two other major families of genetically encoded silencing proteins, proton pumps and anion channels, extended the repertoire of optogenetic tools. Here, we describe the functional and structural characteristics of halorhodopsins. We will discuss the data in light of common principles underlying the mechanism of ion pumps and sensors and will review biophysical and biochemical aspects of neuronal silencers.

Inward H+ pump xenorhodopsin: Mechanism and alternative optogenetic approach.

Generation of an electrochemical proton gradient is the first step of cell bioenergetics. In prokaryotes, the gradient is created by outward membrane protein proton pumps. Inward plasma membrane native proton pumps are yet unknown. We describe comprehensive functional studies of the representatives of the yet noncharacterized xenorhodopsins from Nanohaloarchaea family of microbial rhodopsins. They are inward proton pumps as we demonstrate in model membrane systems, Escherichia coli cells, human embryonic kidney cells, neuroblastoma cells, and rat hippocampal neuronal cells. We also solved the structure of a xenorhodopsin from the nanohalosarchaeon Nanosalina (NsXeR) and suggest a mechanism of inward proton pumping. We demonstrate that the NsXeR is a powerful pump, which is able to elicit action potentials in rat hippocampal neuronal cells up to their maximal intrinsic firing frequency. Hence, inwardly directed proton pumps are suitable for light-induced remote control of neurons, and they are an alternative to the well-known cation-selective channelrhodopsins.

Structural and biochemical studies of sulphotransferase 18 from Arabidopsis thaliana explain its substrate specificity and reaction mechanism.

Sulphotransferases are a diverse group of enzymes catalysing the transfer of a sulfuryl group from 3'-phosphoadenosine 5'-phosphosulphate (PAPS) to a broad range of secondary metabolites. They exist in all kingdoms of life. In Arabidopsis thaliana (L.) Heynh. twenty-two sulphotransferase (SOT) isoforms were identified. Three of those are involved in glucosinolate (Gl) biosynthesis, glycosylated sulphur-containing aldoximes containing chemically different side chains, whose break-down products are involved in stress response against herbivores, pathogens, and abiotic stress. To explain the differences in substrate specificity of desulpho (ds)-Gl SOTs and to understand the reaction mechanism of plant SOTs, we determined the first high-resolution crystal structure of the plant ds-Gl SOT AtSOT18 in complex with 3'-phosphoadenosine 5'-phosphate (PAP) alone and together with the Gl sinigrin. These new structural insights into the determination of substrate specificity were complemented by mutagenesis studies. The structure of AtSOT18 invigorates the similarity between plant and mammalian sulphotransferases, which illustrates the evolutionary conservation of this multifunctional enzyme family. We identified the essential residues for substrate binding and catalysis and demonstrated that the catalytic mechanism is conserved between human and plant enzymes. Our study indicates that the loop-gating mechanism is likely to be a source of the substrate specificity in plants.

Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.

Total chemical synthesis of a membrane protein domain analogue containing two transmembrane helices: functional reconstitution of the semisynthetic sensory rhodopsin/transducer complex.

Negative phototaxis in Archaea is mediated by the sensory rhodopsin II/transducer complex (NpSRII/NpHtrII). After light excitation, the signal is relayed from the receptor to NpHtrII where a rotary motion of TM2 in the membrane domain (NpHtrII1-114) is induced. This conformational change is transferred to the downstream two-component signaling cascade. Here, we describe the chemical synthesis of this membrane domain, which consists of the two transmembrane helices TM1 and TM2. NpHtrII1-114 was synthesized using two sequential ligation steps. The first ligation between NpHtrII47-59 and NpHtrII60-114 was performed in organic solvents, whereas the final ligation was successful in an aqueous buffer that contained a detergent and a denaturant. The product was refolded into micelles and showed functional properties as determined by binding studies to its cognate receptor NpSRII and by photocycle experiments. This work demonstrates that membrane proteins can be successfully synthesized by chemical means paving the way for tailor-made modifications.

Kinetic and thermodynamic analysis of the light-induced processes in plant and cyanobacterial phytochromes.

The light-induced processes of the biological photoreceptor phytochrome (recombinant phyA of oat and recombinant CphA from the cyanobacterium Tolypothrix PCC7601) have been investigated in a time-resolved manner in the temperature range from 0 to 30°C. Both proteins were heterologously expressed and assembled in vitro with phycocyanobilin. The Pr state of plant phytochrome phyA is converted to the Pfr state after formation of four intermediates with an overall quantum yield of ~18%. The reversal reaction (Pfr-to-Pr) shows several intermediates, all of which, even the first detectable one, exhibit already all spectral features of the Pr state. The canonical phytochrome CphA from Tolypothrix showed a similar intermediate sequence as its plant ortholog. Whereas the kinetics for the forward reaction (Pr-to-Pfr) was nearly identical for both proteins, the reverse process (Pr formation) in the cyanobacterial phytochrome was slower by a factor of three. As found for the Pfr-to-Pr intermediates in the plant protein, also in CphA all detectable intermediates showed the spectral features of the Pr form. For both phytochromes, activation parameters for both the forward and the backward reaction pathways were determined.

Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic ß- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic ß-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by ß-actin and myosin-7A by γ-actin. Our results indicate that ß- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

Global fit analysis of myosin-5b motility reveals thermodynamics of Mg2+-sensitive acto-myosin-ADP states.

Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg(2+)-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg(2+)-ions. Free Mg(2+)-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg(2+)-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg(2+)-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg(2+)-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔH(Mg) and ΔS(Mg)) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg(2+)D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1(‡) and ΔH2(‡)). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.

Cofilin cooperates with fascin to disassemble filopodial actin filaments.

Cells use a large repertoire of proteins to remodel the actin cytoskeleton. Depending on the proteins involved, F-actin is organized in specialized protrusions such as lamellipodia or filopodia, which serve diverse functions in cell migration and sensing. Although factors responsible for directed filament assembly in filopodia have been extensively characterized, the mechanisms of filament disassembly in these structures are mostly unknown. We investigated how the actin-depolymerizing factor cofilin-1 affects the dynamics of fascincrosslinked actin filaments in vitro and in live cells. By multicolor total internal reflection fluorescence microscopy and fluorimetric assays, we found that cofilin-mediated severing is enhanced in fascin-crosslinked bundles compared with isolated filaments, and that fascin and cofilin act synergistically in filament severing. Immunolabeling experiments demonstrated for the first time that besides its known localization in lamellipodia and membrane ruffles, endogenous cofilin can also accumulate in the tips and shafts of filopodia. Live-cell imaging of fluorescently tagged proteins revealed that cofilin is specifically targeted to filopodia upon stalling of protrusion and during their retraction. Subsequent electron tomography established filopodial actin filament and/or bundle fragmentation to precisely correlate with cofilin accumulation. These results identify a new mechanism of filopodium disassembly involving both fascin and cofilin.