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International audience; 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.

Microbial rhodopsins appeared to be the most abundant light-harvesting proteins on the Earth and are the major contributes to the solar energy captured in the sea. They possess highly diverse biological functions. Explosion of research on microbial rhodopsins led to breakthroughs in their applications, in particular, in neuroscience.An unexpected new discovery was a Na+-pumping KR2 rhodopsin from Krokinobacter eikastus, the first light-driven non-proton cation pump. A fundamental difference between proton and other cation pumps is that non-proton pumps cannot use tunneling or Grotthuss mechanism for the ion translocation and, therefore, Na+ pumping cannot be understood in the framework of classical proton pump, like bacteriorhodopsin. Extensive studies on the molecular mechanism of KR2 failed to reveal mechanism of pumping. The existing high-resolution structures relate only to the ground state of the protein and revealed no Na+ inside the protein, which is unusual for active ion transporters.KR2 is only known non proton cation transporter with demonstrated remarkable potential for optogenetic applications and, therefore, elucidation of the mechanism of cation transport is important. To understand conception of cation pumping we solved crystal structures of the functionally key O-intermediate state of physiologically relevant pentameric form of KR2 and its D116N and H30A key mutants at high resolution and performed additional functional studies.The structure of the O-state reveals a sodium ion near the retinal Schiff base coordinated by N112 and D116 residues of the characteristic (for the whole family) NDQ triad. The structural and functional data show that cation uptake and release are driven by a switching mechanism. Surprisely, Na+ pathway in KR2 is lined with the chain of polar pores/cavities, similarly to the channelrhodopsin-2. Using Parinello fast molecular dynamics approach we obtained a molecular movie of a probable ion release.Our data provides insight into the mechanism of a non-proton cation light-driven pumping, strongly suggest close relation of sodium pumps to channel rhodopsins and, we believe, expand the present knowledge of rhodopsin world. Certainly they might be used for engineering of cation pumps and ion channels for optogenetics.

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 preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. 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.

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., Nucleocytoplasmic Large DNA Viruses (NCLDV) that infect algae encode two distinct families of microbial rhodopsins. Here, the authors characterise two proteins form the viral rhodopsin group 1 OLPVR1 and VirChR1, present the 1.4 Å crystal structure of OLPVR1 and show that viral rhodopsins 1 are light-gated cation channels.

Single molecule localization microscopy (SMLM) allows the imaging of cellular structures with resolutions five to ten times below the diffraction limit of optical microscopy. It was originally introduced as a two-dimensional technique based on the localization of single emitters as projection onto the x-y imaging plane. The determination of the axial position of a fluorescent emitter is only possible by additional information. Here we report a method (spatial filter SMLM (SFSMLM)) that allows to determine the axial positions of fluorescent molecules and nanoparticles on the nanometer scale by the usage of two spatial filters, which are placed in two otherwise identical emission detection channels. SFSMLM allows axial localization in a range of ca. 1.5 μm with a localization precision of 15 - 30 nm in axial direction. The technique was utilized for localizing and imaging small cellular structures - e.g. actin filaments, vesicles and mitochondria - in three dimensions.

Rhodopsins are the most abundant light-harvesting proteins. A new family of rhodopsins, heliorhodopsins (HeRs), was recently discovered. In opposite to the known rhodopsins their N-termini face the cytoplasm. HeRs structure and function remain unknown. We present structures of two HeR-48C12 states at 1.5 Å showing its remarkable difference from all known rhodopsins. Its internal extracellular part is completely hydrophobic, while the cytoplasmic part comprises a cavity (’active site’), 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 triangle molecule (acetate) is present in the ‘active site’ which demonstrated its ability to maintain such anions as carbonate or nitrate. 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 into fundamental redox biological processes.

Super-resolution imaging based on single molecule localization of cellular structures on nanometer scale requires to record a series of wide-field or TIRF images resulting in a considerable recording time (typically of minutes). Therefore, sample drift becomes a critical problem and will lower the imaging precision. Herein we utilized morphological features of the specimen (mammalian cells) itself as reference markers replacing the traditionally used markers (e.g., artificial fiduciary markers, fluorescent beads, or metal nanoparticles) for sample drift compensation. We achieved sub-nanometer localization precision

High resolution structures reveal the mechanisms of sodium, potassium, and proton pumping by a light-driven microbial rhodopsin., Rhodopsins are the most universal biological light-energy transducers and abundant phototrophic mechanisms that evolved on Earth and have a remarkable diversity and potential for biotechnological applications. Recently, the first sodium-pumping rhodopsin KR2 from Krokinobacter eikastus was discovered and characterized. However, the existing structures of KR2 are contradictory, and the mechanism of Na+ pumping is not yet understood. Here, we present a structure of the cationic (non H+) light-driven pump at physiological pH in its pentameric form. We also present 13 atomic structures and functional data on the KR2 and its mutants, including potassium pumps, which show that oligomerization of the microbial rhodopsin is obligatory for its biological function. The studies reveal the structure of KR2 at nonphysiological low pH where it acts as a proton pump. The structure provides new insights into the mechanisms of microbial rhodopsins and opens the way to a rational design of novel cation pumps for optogenetics.

Since the introduction of in meso crystallization of membrane proteins in lipidic cubic phase (LCP) by Landau and Rosenbusch in 1996, numerous studies attempted to elucidate the mechanism of in meso crystal nucleation and growth. Here we present a fluorescence microscopy study of the crystallization process of the light-driven proton pump bacteriorhodopsin. The crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. These observations suggest an Ostwald ripening mechanism of the in meso crystal growth. Analysis of the microcrystal spatial distribution suggests that microcrystal nucleation occurs predominately at the LCP domain boundaries.

Susceptibility to fungal infection is commonly associated with impaired neutrophil responses. To study the mechanisms underlying this association, we investigated neutrophil recruitment to the conducting airway wall afterAspergillus fumigatusconidium inhalation in mouse models of drug-induced immunosuppression and antibody-mediated neutrophil depletion (neutropenia) by performing three-dimensional confocal laser-scanning microscopy of whole-mount primary bronchus specimens. Actin staining enabled visualization of the epithelial and smooth muscle layers that mark the airway wall. Gr-1+or Ly6G+neutrophils located between the epithelium and smooth muscles were considered airway wall neutrophils. The number of airway wall neutrophils for immunocompetent, immunosuppressed, and neutropenic mice before and 6 h afterA. fumigatusinfection were analyzed and compared. Our results show that the number of conducting airway wall neutrophils in immunocompetent mice significantly increased upon inflammation, while a dramatic reduction in this number was observed following immunosuppression and neutropenia. Interestingly, a slight increase in the infiltration of neutrophils into the airway wall was detected as a result of infection, even in immunosuppressed and neutropenic mice. Taken together, these data indicate that neutrophils are present in intact conducting airway walls and the number elevates uponA. fumigatusinfection. Conducting airway wall neutrophils are affected by both neutropenia and immunosuppression.

Research on halophilic microorganisms is important due to their relation to fundamental questions of survival of living organisms in a hostile environment. Here we introduce a novel method to stain halophiles with MitoTracker fluorescent dyes in their growth medium. The method is based on membrane-potential sensitive dyes, which were originally used to label mitochondria in eukaryotic cells. We demonstrate that these fluorescent dyes provide high staining efficiency and are beneficial for multi-staining purposes due to the spectral range covered (from orange to deep red). In contrast with other fluorescent dyes used so far, MitoTracker does not affect growth rate, and remains in cells after several washing steps and several generations in cell culture. The suggested dyes were tested on three archaeal (Hbt. salinarum, Haloferax sp., Halorubrum sp.) and two bacterial (Salicola sp., Halomonas sp.) strains of halophilic microorganisms. The new staining approach provides new insights into biology of Hbt. salinarum. We demonstrated the interconversion of rod-shaped cells of Hbt. salinarium to spheroplasts and submicron-sized spheres, as well as the cytoplasmic integrity of giant rod Hbt. salinarum species. By expanding the variety of tools available for halophile detection, MitoTracker dyes overcome long-standing limitations in fluorescence microscopy studies of halophiles.

INTRODUCTION Ion channels are integral membrane proteins that upon stimulation modulate the flow of ions across the cell or organelle membrane. The resulting electrical signals are involved in biological functions such as electrochemical transmission and information processing in neurons. Channelrhodopsins (ChRs) appear to be unusual channels. They belong to the large family of microbial rhodopsins, seven-helical transmembrane proteins containing retinal as chromophore. Photon absorption initiates retinal isomerization resulting in a photocycle, with different spectroscopically distinguishable intermediates, thereby controlling the opening and closing of the channel. In 2003, it was demonstrated that light-induced currents by heterologously expressed ChR2 can be used to change a host’s membrane potential. The concept was further applied to precisely control muscle and neural activity by using light-induced depolarization to trigger an action potential in neurons expressing ChR2. This optogenetic approach with ChR2 and other ChRs has been widely used for remote control of neural cells in culture and in living animals with high spatiotemporal resolution. It is also used in biomedical studies aimed to cure severe diseases. RATIONALE Despite the wealth of biochemical and biophysical data, a high-resolution structure and structural mechanisms of a native ChR2 (and other ChRs) have not yet been known. A step forward was the structure of a chimera (C1C2). However, recent electrophysiological and Fourier transform infrared data showed that C1C2 exhibits light-induced responses that are functionally and mechanistically different from ChR2. Given that ChR2 is the most frequently used tool in optogenetics, a high-resolution structure of ChR2 is of high importance. Deciphering the structure of the native channel would shed light on how the light-induced changes at the retinal Schiff base (RSB) are linked to the channel operation and may make engineering of enhanced optogenetic tools more efficient. RESULTS We expressed ChR2 in LEXSY and used in the meso crystallization approach to determine the crystal structure of the wild-type ChR2 and C128T slow mutant at 2.4 and 2.7 A, respectively (C, cysteine; T, threonine). Two different dark-state conformations of ChR2 in the two protomers in the asymmetric unit were resolved. The overall structure alignment of the protomers does not show a visible difference in backbone conformation. However, the conformation of some amino acids and the position of water molecules are not the same. The dimerization is strong and provided mainly through the interaction of helices 3 and 4 and the N termini. In addition, the protomers are connected with a disulfide bond, C34/C36′. In both protomers, we identified ion conduction pathway comprising four cavities [extracellular cavity 1 (EC1), EC2, intracellular cavity 1 (IC1), and IC2] that are separated by three gates [extracellular gate (ECG), central gate (CG), and intracellular gate (ICG)] (figure, panel A). Arginines R120 and R268 are the cores of ECG and ICG, respectively, in all ChRs. The Schiff base is hydrogen-bond–connected to E123 and D253 amino acids (E, glutamic acid; D, aspartic acid) and is a key part of the CG that is further connected with two other gates through an extended H-bond network mediated by numerous water molecules (figure, panel B). The DC gate is separate from the gates in the channel pathway and is bridged by hydrogen bonds through the water molecule w5. Hydrogen bonding of the DC pair (C128 and D156) has two important consequences. It stabilizes helices 3 and 4 and provides connection from D156, a possible proton donor, to the RSB. The presence of the hydrogen bonds provides structural insights into how the DC gate controls ChR2 gating lifetime. CONCLUSION The determined structures of ChR2 and its C128T mutant present the molecular basis for the understanding of ChR functioning. They provide insights into mechanisms of channel opening and closing. A plausible scenario is that the disruption of the H-bonds between E123 and D253 and the Schiff base and the protonation of D253 upon retinal isomerization trigger rearrangements in the extended hydrogen-bonded networks, stabilizing the ECG and CG and also rearranging the H-bonding network in the cavities. Upon retinal isomerization, these two gates are opened and the network is broken. This leads to the reorientation of helix 2. Additional changes in helices 6 and 7 induced by the isomerization could help with opening the ICG and channel pore formation.

Protein biosynthesis is inherently coupled to cotranslational protein folding. Folding of the nascent chain already occurs during synthesis and is mediated by spatial constraints imposed by the ribosomal exit tunnel as well as self-interactions. The polypeptide's vectorial emergence from the ribosomal tunnel establishes the possible folding pathways leading to its native tertiary structure. How cotranslational protein folding and the rate of synthesis are linked to a protein's amino acid sequence is still not well defined. Here, we follow synthesis by individual ribosomes using dual-trap optical tweezers and observe simultaneous folding of the nascent polypeptide chain in real time. We show that observed stalling during translation correlates with slowed peptide bond formation at successive proline sequence positions and electrostatic interactions between positively charged amino acids and the ribosomal tunnel. We also determine possible cotranslational folding sites initiated by hydrophobic collapse for an unstructured and two globular proteins while directly measuring initial cotranslational folding forces. Our study elucidates the intricate relationship among a protein's amino acid sequence, its cotranslational nascent-chain elongation rate, and folding.

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.

Membrane-like nanodiscs (ND) have become an important tool for the cell-free expression, solubilization, folding, and in vitro structural and functional studies of membrane proteins (MPs). Direct crystallization of MPs embedded in NDs would be of high importance for structural biology. However, despite considerable efforts we have been as yet unable to obtain crystals suitable for X-ray crystallography. In the present work, we show that an ND-trapped MP can be transferred into the cubic phase and crystallized in meso. Bacteriorhodopsin (BR) reconstituted into nanodiscs was mixed with a lipidic mesophase and crystallization was induced by adding a precipitant. The resulting crystals diffract beyond 1.8 A. The structure of BR was solved at 1.9 A and found to be indistinguishable from previous structures obtained with the protein solubilized in detergent. We suggest the proposed protocol of in meso crystallization to be generally applicable to ND-trapped MPs.

One of the major and essential classes of transmembrane (TM) receptors, present in all domains of life, is sensor histidine kinases, parts of two-component signaling systems (TCSs). The structural mechanisms of TM signaling by these sensors are poorly understood. We present crystal structures of the periplasmic sensor domain, the TM domain, and the cytoplasmic HAMP domain of the

The method of small angle scattering (SAS) is widely used in the field of biophysical research of proteins in aqueous solutions. Obtaining low-resolution structure of proteins is still a highly valuable method despite the advances in high-resolution methods such as X-ray diffraction, cryo-EM etc. SAS offers the unique possibility to obtain structural information under conditions close to those of functional assays, i.e. in solution, without different additives, in the mg/mL concentration range. SAS method has a long history, but there are still many uncertainties related to data treatment. We compared 1D SAS profiles of apoferritin obtained by X-ray diffraction (XRD) and SAS methods. It is shown that SAS curves for X-ray diffraction crystallographic structure of apoferritin differ more significantly than it might be expected due to the resolution of the SAS instrument. Extrapolation to infinite dilution (EID) method does not sufficiently exclude dimerization and oligomerization effects and therefore could not guarantee total absence of dimers account in the final SAS curve. In this study, we show that EID SAXS, EID SANS and SEC-SAXS methods give complementary results and when they are used all together, it allows obtaining the most accurate results and high confidence from SAS data analysis of proteins.

Recently, the first known light-driven sodium pumps, from the microbial rhodopsin family, were discovered. We have solved the structure of one of them, Krokinobacter eikastus rhodopsin 2 (KR2), in the monomeric blue state and in two pentameric red states, at resolutions of 1.45 A and 2.2 and 2.8 A, respectively. The structures reveal the ion-translocation pathway and show that the sodium ion is bound outside the protein at the oligomerization interface, that the ion-release cavity is capped by a unique N-terminal α-helix and that the ion-uptake cavity is unexpectedly large and open to the surface. Obstruction of the cavity with the mutation G263F imparts KR2 with the ability to pump potassium. These results pave the way for the understanding and rational design of cation pumps with new specific properties valuable for optogenetics.

Protein synthesis is directly linked to protein folding. Translation kinetics plays a crucial role in forming secondary structural elements that lead to tertiary structure formation. However, how these two processes are coordinated is still not fully understood.Single-molecule techniques are essential for the study of protein synthesis and subsequent folding and were used in the past (1), since these are asynchronous processes that are extremely difficult to be observed using ensemble methods.Here we show for the first time to our knowledge full synthesis of three distinct proteins (hTau40, DHFR and GFPem) studied with dual-trap optical tweezers at the single-molecule level, continuing our previously published work (2). Our study explicates the intricate relationship between a protein's amino acid sequence, its co-translational elongation rate and folding.We show how hydrophobic collapse, which is the first step in protein folding, correlates with the number of hydrophobic amino acids that emerge from the ribosomal tunnel during synthesis. We also find that translational pauses are caused mainly by consecutive proline residues and positively charged amino acids. Finally, we propose a model that predicts the translation rate at a certain applied force and estimates hydrophobic collapse nucleation sites (manuscript submitted).1.Katranidis, A., D. Atta, R. Schlesinger, K. H. Nierhaus, T. Choli-Papadopoulou, I. Gregor, M. Gerrits, G. Buldt, and J. Fitter. 2009. Angew. Chem. Intl. ed 48, 1758-17612.Katranidis, A., W. Grange, R. Schlesinger, T. Choli-Papadopoulou, D. Bruggemann, M. Hegner, and G. Buldt. 2011. FEBS Lett. 585, 1859-1863