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The ability to cope with and adapt to changes in the environment is essential for all organisms. Osmotic pressure is a universal threat when environmental changes result in an imbalance of osmolytes inside and outside the cell which causes a deviation from the normal turgor. Cells have developed a potent system to deal with this stress in the form of mechanosensitive ion channels. Channel opening releases solutes from the cell and relieves the stress immediately. In bacteria, these channels directly sense the increased membrane tension caused by the enhanced turgor levels upon hypoosmotic shock. The mechanosensitive channel of small conductance, MscS, from Escherichia coli is one of the most extensively studied examples of mechanically stimulated channels. Different conformational states of this channel were obtained in various detergents and membrane mimetics, highlighting an intimate connection between the channel and its lipidic environment. Associated lipids occupy distinct locations and determine the conformational states of MscS. Not all these features are preserved in the larger MscS-like homologues. Recent structures of homologues from bacteria and plants identify common features and differences. This review discusses the current structural and functional models for MscS opening, as well as the influence of certain membrane characteristics on gating. [ABSTRACT FROM AUTHOR]

The bacterial mechanosensitive channel of small conductance (MscS) is a well-studied model of how mechanical forces from the membrane can be sensed by an embedded protein. A recent study by Zhang et al. visualizes how MscS behaves under membrane tension, entering a desensitized state when it loses all coordinated lipids. [ABSTRACT FROM AUTHOR]

Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation. MscS senses the increase of tension in the membrane directly by the force from the lipids, but the molecular mechanism is still elusive. We determined the lipid interactions of MscS by resolving the structure of Escherichia coli MscS embedded in membrane discs to 2.9-Å resolution using cryo-electron microscopy. The membrane is attached only to parts of the sensor paddles of MscS, but phospholipid molecules move through grooves into remote pockets on the cytosolic side. On the periplasmic side, a lipid bound by R88 at the pore entrance is separated from the membrane by TM1 helices. The N-terminus interacts with the periplasmic membrane surface. We demonstrate that the unique membrane domain of MscS promotes deep penetration of lipid molecules and shows multimodal interaction with the membrane to fine-tune tension sensing. • MscS, a mechanosensitive channel, opens in response to high tension in the membrane • The first structure of MscS within the membrane was obtained by cryo-EM to 2.9 Å • The paddles of MscS are only partly embedded in the membrane • 21 lipid molecules are resolved within the complex, some coordinated by R59 and R88 • Tension sensing relies on these molecular interactions of MscS with lipids Unlabelled Image [ABSTRACT FROM AUTHOR]

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains. Here, we analyze protein–lipid interactions for MscS by quenching of tryptophan fluorescence with brominated lipids. By this strategy, we define the limits of the bilayer for TM1, which is the most lipid exposed helix of this protein. In addition, we show that residues deep in the pockets, created by the oligomeric assembly, interact with lipid chains. On the cytoplasmic side, lipids penetrate as far as the pore-lining helices and lipid molecules can align along TM3b perpendicular to lipids in the bilayer. Cardiolipin, free fatty acids, and branched lipids can access the pockets where the latter have a distinct effect on function. Cholesterol is excluded from the pockets. We demonstrate that introduction of hydrophilic residues into TM3b severely impairs channel function and that even "conservative" hydrophobic substitutions can modulate the stability of the open pore. The data provide important insights into the interactions between phospholipids and MscS and are discussed in the light of recent developments in the study of Piezo1 and TrpV4. Unlabelled Image • MscS tension-sensing relies on dynamic molecular interactions with lipids. • Branched lipids or protein mutations that change these interactions modify gating. • Lipids can penetrate in pockets deep within MscS by changing orientation. • Tryptophan fluorescence quenching is defined by bilayer lipid interactions with MscS. • MscS deforms the shape of the membrane in a manner similar to Piezo1. [ABSTRACT FROM AUTHOR]

Mechanosensitive (MS) channels provide protection against hypo-osmotic shock in bacteria whereas eukaryotic MS channels fulfil a multitude of important functions beside osmoregulation. Interactions with the membrane lipids are responsible for the sensing of mechanical force for most known MS channels. It emerged recently that not only prokaryotic, but also eukaryotic, MS channels are able to directly sense the tension in the membrane bilayer without any additional cofactor. If the membrane is solely viewed as a continuous medium with specific anisotropic physical properties, the sensitivity towards tension changes can be explained as result of the hydrophobic coupling between membrane and transmembrane (TM) regions of the channel. The increased cross-sectional area of the MS channel in the active conformation and elastic deformations of the membrane close to the channel have been described as important factors. However, recent studies suggest that molecular interactions of lipids with the channels could play an important role in mechanosensation. Pockets in between TM helices were identified in the MS channel of small conductance (MscS) and YnaI that are filled with lipids. Less lipids are present in the open state of MscS than the closed according to MD simulations. Thus it was suggested that exclusion of lipid fatty acyl chains from these pockets, as a consequence of increased tension, would trigger gating. Similarly, in the eukaryotic MS channel TRAAK it was found that a lipid chain blocks the conducting path in the closed state. The role of these specific lipid interactions in mechanosensation are highlighted in this review. [ABSTRACT FROM AUTHOR]

Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli and Gram-negative bacteria that live in the human gut must be able to tolerate rapid and large changes in environmental pH. Low pH irreversibly denatures and precipitates many bacterial proteins. While cytoplasmic proteins are well buffered against such swings, periplasmic proteins are not. Instead, it appears that some bacteria utilize chaperone proteins that stabilize periplasmic proteins, preventing their precipitation. Two highly expressed and related proteins, HdeA and HdeB, have been identified as acid-activated chaperones. The structure of HdeA is known and a mechanism for activation has been proposed. In this model, dimeric HdeA dissociates at low pH, and the exposed dimeric interface binds exposed hydrophobic surfaces of acid-denatured proteins, preventing their irreversible aggregation. We now report the structure and biophysical characterization of the HdeB protein. The monomer of HdeB shares a similar structure with HdeA, but its dimeric interface is different in composition and spatial location. We have used fluorescence to study the behavior of HdeB as pH is lowered, and like HdeA, it dissociates to monomers. We have identified one of the key intersubunit interactions that controls pH-induced monomerization. Our analysis identifies a structural interaction within the HdeB monomer that is disrupted as pH is lowered, leading to enhanced structural flexibility. [Copyright &y& Elsevier]

Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL. Consequently, in cells, gating has severe consequences for energetics and homoeostasis, since membrane depolarization and modification of cytoplasmic ionic composition is an immediate consequence. Protection against disruption of cellular integrity, which is the function of the major channels, provides a strong evolutionary rationale for possession of such disruptive channels. The elegant crystal structures for these channels has opened the way to detailed investigations that combine molecular genetics with electrophysiology and studies of cellular behaviour. In the present article, the focus is primarily on the structure of MscS, the small mechanosensitive channel. The description of the structure is accompanied by discussion of the major sites of channel–lipid interaction and reasoned, but limited, speculation on the potential mechanisms of tension sensing leading to gating. [ABSTRACT FROM AUTHOR]

Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions. We show that Trp can be inserted in place of Leu at the two pore seal positions, Leu105 and Leo109, resulting in functional channels. Using Trp105 as a probe, we demonstrate that the A106V mutation causes a modified conformation in the purified channel protein consistent with a more open state in solution. Moreover, we show that solubilized MscS changes to a more open conformation in the presence of phospholipids or their lysolorms. [ABSTRACT FROM AUTHOR]

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels. Three Trp residues occur naturally in the Escherichia coli MscS (MscS-Ec) protein: W16 lies in the periplasm, immediately before the first transmembrane span (TM1), whereas W240 and W251 lie at the subunit interfaces that create the cytoplasmic vestibule portals. The role of these residues in MscS function and stability were investigated using site-directed mutagenesis. Functional channels with altered properties were created when any of the Trp residues were replaced by another amino acid, with the greatest retention of function associated with pheriylalanine (Phe) substitutions. Analysis of the fluorescence properties of purified mutant MscS proteins containing single Trp residues revealed that W16 and W251 are relatively inaccessible, whereas W240 is accessible to quenching agents. The data point to a significant role for W16 in the gating of MscS, and an essential role for W240 in MscS oligomer stability. [ABSTRACT FROM AUTHOR]

Identifies two tetranuclear FeS clusters on the ferredoxin-type subunit of a NADH:ubiquinone oxireductase or complex I from Neurospora crassa and Escherichia coli. Ultraviolet/visible and electron paramagnetic resonance spectroscopy; Reaction of NADH-reduced complex I with oxygen; Redox titration of the redox group.

Identifies Cu[sub z], the catalytic center in nitrous oxide reductase as a copper--sulfide cluster. Application of Raman spectroscopy in the study; Isolation of the reductase from the bacteria Paracoccus pantotrophus and Pseudomonas stutzeri; Existence of an acid-labile sulfur ligand in the catalytic center.

20 examples [ABSTRACT FROM AUTHOR]

[Display omitted] Many drugs and drug candidates are poorly water-soluble. Intestinal fluids play an important role in their solubilization. However, the interactions of intestinal fluids with polymer excipients, drugs and their formulations are not fully understood. Here, diffusion ordered spectroscopy (DOSY) and nuclear Overhauser effect spectroscopy (NOESY), complemented by cryo-TEM were employed to address this. Efavirenz (EFV) as model drug, the triblock copolymers Pluronic® F-127 (PF127) and poly(2-oxazoline) based pMeOx- b -pPrOzi- b -pMeOx (pOx/pOzi) and their respective formulations were studied in simulated fed-state intestinal fluid (FeSSIF). For the individual polymers, the bile interfering nature of PF127 was confirmed and pure pOx/pOzi was newly classified as non-interfering. A different and more complex behaviour was however observed if EFV was involved. PF127/EFV formulations in FeSSIF showed concentration dependent aggregation with separate colloids at low formulation concentrations, a merging of individual particles at the solubility limit of EFV in FeSSIF and joint aggregates above this concentration. In the case of pOx/pOzi/EFV formulations, coincident diffusion coefficients for pOx/pOzi, lipids and EFV indicate joint aggregates across the studied concentration range. This demonstrates that separate evaluation of polymers and drugs in biorelevant media is not sufficient and their mixtures need to be studied to learn about concentration and composition dependent behaviour. [ABSTRACT FROM AUTHOR]

We describe a mechanosensitive (MS) channel that has mechanosensitive channel of miniconductance (MscM) activity, and displays unique properties with respect to gating. Mechanosensitive channels respond to membrane tension, are ubiquitous from bacteria to man, and exhibit a great diversity in structure and function. These channels protect Bacteria and Archaea against hypoosmotic shock and are critical determinants of shape in chloroplasts. Given the dominant roles played in bacteria by the mechanosensitive channel of small conductance (MscS) and the mechanosensitive channel of large conductance (MscL), the role of the multiple MS channel homologs observed in most organisms remains obscure. Here we demonstrate that a MscS homolog, YbdG, extends the range of hypoosmotic shock that Escherichia coli cells can survive, but its expression level is insufficient to protect against severe shocks. Overexpression of the YbdG protein provides complete protection. Transcription and translation of the ybdG gene are enhanced by osmotic stress consistent with a role for the protein in survival of hypoosmotic shock. Measurement of the conductance of the native channel by standard patch clamp methods was not possible. However, a fully functional YbdG mutant channel, V229A, exhibits a conductance in membrane patches consistent with MscM activity. We find that MscM activities arise from more than one gene product because ybdG deletion mutants still exhibit an occasional MscM-like conductance. We propose that ybdG encodes a low-abundance MscM-type MS channel, which in cells relieves low levels of membrane tension, obviating the need to activate the major MS channels, MscS and MscL. [ABSTRACT FROM AUTHOR]

The mechanosensitive channel of small conductance (MscS) protects bacteria against hypoosmotic shock. It can sense the tension in the surrounding membrane and releases solutes if the pressure in the cell is getting too high. The membrane contacts MscS at sensor paddles, but lipids also leave the membrane and move along grooves between the paddles to reside as far as 15 Å away from the membrane in hydrophobic pockets. One sensing model suggests that a higher tension pulls lipids from the grooves back to the membrane, which triggers gating. However, it is still unclear to what degree this model accounts for sensing and what contribution the direct interaction of the membrane with the channel has. Here, we show that MscS opens when it is sufficiently delipidated by incubation with the detergent dodecyl-β-maltoside or the branched detergent lauryl maltose neopentyl glycol. After addition of detergent-solubilized lipids, it closes again. These results support the model that lipid extrusion causes gating: Lipids are slowly removed from the grooves and pockets by the incubation with detergent, which triggers opening. Addition of lipids in micelles allows lipids to migrate back into the pockets, which closes the channel even in the absence of a membrane. Based on the distribution of the aliphatic chains in the open and closed conformation, we propose that during gating, lipids leave the complex on the cytosolic leaflet at the height of highest lateral tension, while on the periplasmic side, lipids flow into gaps, which open between transmembrane helices. [ABSTRACT FROM AUTHOR]

Reduced inorganic sulfur compounds are utilized by many bacteria as electron donors to photosynthetic or respiratory electron transport chains. This metabolism is a key component of the biogeochemical sulfur cycle. The SoxAX protein is a heterodimeric c-type cytochrome involved in thiosulfate oxidation. The crystal structures of SoxAX from the photosynthetic bacterium Rhodovulum sulfidophilum have been solved at 1.75 A resolution in the oxidized state and at 1,5 A resolution in the dithionite-reduced state, providing the first structural insights into the enzymatic oxidation of thiosulfate. The SoxAX active site contains a haem with unprecedented cysteine persulfide (cysteine sulfane) coordination. This unusual post- translational modification is also seen in sulfurtransferases such as rhodanese. Intriguingly, this enzyme shares further active site characteristics with SoxAX such as an adjacent conserved arginine residue and a strongly positive electrostatic potential. These similarities have allowed us to suggest a catalytic mechanism for enzymatic thiosulfate oxidation. The atomic coordinates and experimental structure factors have been deposited in the PDB with the accession codes 1H31, 1H32 and 1H33. [ABSTRACT FROM AUTHOR]

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that finetunes osmoregulation but is likely to fulfill additional functions. Escherichia coli has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood. The medium-sized channel YnaI was extracted and delivered in native nanodiscs in closed-like and open-like conformations using the copolymer diisobutylene/maleic acid (DIBMA) for structural studies. Here we show by electron cryomicroscopy that YnaI has an extended sensor paddle that during gating relocates relative to the pore concomitant with bending of a GGxGG motif in the pore helices. YnaI is the only one of the six paralogs that has this GGxGG motif allowing the sensor paddle to move outward. Access to the pore is through a vestibule on the cytosolic side that is fenestrated by side portals. In YnaI, these portals are obstructed by aromatic side chains but are still fully hydrated and thus support conductance. For comparison with large-sized channels, we determined the structure of YbiO, which showed larger portals and a wider pore with no GGxGG motif. Further in silico comparison of MscS, YnaI, and YbiO highlighted differences in the hydrophobicity and wettability of their pores and vestibule interiors. Thus, MscS-like channels of different sizes have a common core architecture but show different gating mechanisms and fine-tuned conductive properties. [ABSTRACT FROM AUTHOR]

Highlights • The Falcon III camera has integrating and counting mode acquisition. • Maps were obtained with resolutions of 97% of Nyquist frequency in the counting mode. • Reconstructions of medium sized complexes (0.5 MDa) gain from the counting mode. • The structure of TMV was determined at 2.3 Å resolution from data collected in integrating mode. Abstract Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e−/Px² and integrating mode capability at exposure rates above 10 e−/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode. [ABSTRACT FROM AUTHOR]

Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype. [ABSTRACT FROM AUTHOR]

Summary Mechanosensitive channels protect bacteria against lysis caused by a sudden drop in osmolarity in their surroundings. Besides the channel of large conductance (MscL) and small conductance (MscS), Escherichia coli has five additional paralogs of MscS that are functional and widespread in the bacterial kingdom. Here, we present the structure of YnaI by cryo-electron microscopy to a resolution of 13 Å. While the cytosolic vestibule is structurally similar to that in MscS, additional density is seen in the transmembrane (TM) region consistent with the presence of two additional TM helices predicted for YnaI. The location of this density suggests that the extra TM helices are tilted, which could induce local membrane curvature extending the tension-sensing paddles seen in MscS. Off-center lipid-accessible cavities are seen that resemble gaps between the sensor paddles in MscS. The conservation of the tapered shape and the cavities in YnaI suggest a mechanism similar to that of MscS. [ABSTRACT FROM AUTHOR]

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-Sconjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of 10-20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S -{[5-(dimethylamino)naphthalen-1-yl] sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N -ethylsuccinimido-S -glutathione, has a 10-20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S -lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system. [ABSTRACT FROM AUTHOR]

Escherichia coli and many other Gram-negative pathogenic bacteria protect themselves from the toxic effects of electrophilic compounds by using a potassium efflux system (Kef). Potassium efflux is coupled to the influx of protons, which lowers the internal pH and results in immediate protection. The activity of the Kef system is subject to complex regulation by glutathione and its S conjugates. Full activation of KefC requires a soluble ancillary protein, KefF. This protein has structural similarities to oxidoreductases, including human quinone reductases 1 and 2. Here, we show that KefF has enzymatic activity as an oxidoreductase, in addition to its role as the KefC activator. It accepts NADH and NADPH as electron donors and quinones and ferriojanide (in addition to other compounds) as acceptors. However, typical electrophilic activators of the Kef system, e.g., N-ethyl maleimide, are not substrates. If the enzymatic activity is disrupted by site-directed mutagenesis while retaining structural integrity, KefF is still able to activate the Kef system, showing that the role as an activator is independent of the enzyme activity. Potassium efflux assays show that electrophilic quinones are able to activate the Kef system by forming S conjugates with glutathione. Therefore, it appears that the enzymatic activity of KefF diminishes the redox toxicity of quinones, in parallel with the protection afforded by activation of the Kef system. [ABSTRACT FROM AUTHOR]

Summary: KTN (RCK) domains are nucleotide-binding folds that form the cytoplasmic regulatory complexes of various K+ channels and transporters. The mechanisms these proteins use to control their transmembrane pore-forming counterparts remains unclear despite numerous electrophysiological and structural studies. KTN (RCK) domains consistently crystallize as dimers within the asymmetric unit, forming a pronounced hinge between two Rossmann folds. We have previously proposed that modification of the hinge angle plays an important role in activating the associated membrane-integrated components of the channel or transporter. Here we report the structure of the C-terminal, KTN-bearing domain of the E. coli KefC K+ efflux system in association with the ancillary subunit, KefF, which is known to stabilize the conductive state. The structure of the complex and functional analysis of KefC variants reveal that control of the conformational flexibility inherent in the KTN dimer hinge is modulated by KefF and essential for regulation of KefC ion flux. [Copyright &y& Elsevier]

Atlantic salmon (Salmo salar L.) smolts (mean weight 66 g) in freshwater were exposed to three replicate levels of carbon dioxide (CO2) partial pressures for 60 days in an open flow system: 2 mm Hg (6 mg l−1; control), 5 mm Hg (16 mg l−1; medium) and 7 mm Hg (24 mg l−1; high) at constant pH. Water temperature was 7–9 °C and oxygen concentration higher than 9 mg l−1. All groups were transferred to 34‰ seawater on day 60 and kept there for 150 days. No significant differences in weight and length were found between groups during the freshwater and seawater period. While mean cellular haemoglobin (in the high group) and plasma chloride (in the medium and high group) were significantly lower than that of the control group during the freshwater period, no significant differences between groups were observed in mean haemoglobin concentration, haematocrit, or gill Na+,-K+-ATPase activity. Increased nephrocalcinosis and contents of Ca in the kidney in response to increasing water CO2 levels were observed at day 58. After 4 weeks in seawater, pH in muscle and whole body ash, Ca, P and Zn content were all significantly higher in fish exposed to elevated CO2 levels during the freshwater period. This indicates that freshwater CO2 levels affected tissue mineral content after transfer to seawater. [Copyright &y& Elsevier]

Provides information on the proton-translocating NADH:ubiquinone oxidoreductase of mitochondria (complex I). Use of diode array UV/VIS spectroscopy under anaerobic conditions; Location of redox states in electron pathway; Characteristics of complex I redox groups.