Genetic assays capable of measuring the propensity of transmembrane helices to oligomerize within the cytoplasmic membrane of the bacterium
are frequently used when sequence-specificity in ...transmembrane helix-helix interactions is investigated. In the present study, dimerization of the well-investigated wild-type and G83I-mutated transmembrane helix of the human glycophorin A protein was studied. Gradual prolongation of the transmembrane helix at the C-terminus with Leu residues lead to pronounced changes in the dimerization propensity when measured with the TOXCAT assay. Thus, besides sequence specificity, hydrophobic mismatch between the hydrophobic core of a studied transmembrane helix and the
membrane can impact the oligomerization propensity of a transmembrane helix. This suggests that the results of genetic assays aiming at determining interactions of heterologous transmembrane helices within the
membrane do not necessarily solely reflect sequence specificity in transmembrane helix-helix interactions, but might be additionally modulated by topological and structural effects caused by hydrophobic mismatch.
The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological ...function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg
-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show
that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.
The function of IM30 (also known as Vipp1) is linked to protection and/or remodeling of the thylakoid membrane system in chloroplasts and cyanobacteria. Recently, it has been revealed that the ...Arabidopsis IM30 protein exhibits GTP hydrolyzing activity in vitro, which was unexpected, as IM30 does not show any classical GTPase features. In the present study, we addressed the question, whether an apparent GTPase activity is conserved in IM30 proteins and can also be observed for IM30 of the cyanobacterium Synechocystis sp. PCC 6803. We show that Synechocystis IM30 is indeed able to bind and hydrolyze GTP followed by the release of P
. Yet, the apparent GTPase activity of Synechocystis IM30 does not depend on Mg
, which, together with the lack of classical GTPase features, renders IM30 an atypical GTPase. To elucidate the impact of this cryptic GTPase activity on the membrane remodeling activity of IM30, we tested whether GTP hydrolysis influences IM30 membrane binding and/or IM30-mediated membrane fusion. We show that membrane remodeling by Synechocystis IM30 is slightly affected by nucleotides. Yet, despite IM30 clearly catalyzing GTP hydrolysis, this does not seem to be vital for its membrane remodeling function.
Biogenesis and dynamics of thylakoid membranes likely involves membrane fusion events. Membrane attachment of the inner membrane-associated protein of 30 kDa (IM30) affects the structure of the lipid ...bilayer, finally resulting in membrane fusion. Yet, how IM30 triggers membrane fusion is largely unclear. IM30 monomers pre-assemble into stable tetrameric building blocks, which further align to form oligomeric ring structures, and differently sized IM30 rings bind to membranes. Based on a 3D reconstruction of IM30 rings, we locate the IM30 loop 2 region at the bottom of the ring and show intact membrane binding but missing fusogenic activity of loop 2 mutants. However, helix 7, which has recently been shown to mediate membrane binding, was located at the oppossite, top side of IM30 rings. We propose that a two-sided IM30 ring complex connects two opposing membranes, finally resulting in membrane fusion. Thus, IM30-mediated membrane fusion requires a Janus-faced IM30 ring.
Display omitted
•IM30 monomers assemble into stable tetrameric building blocks prior to ring formation•3D reconstructions of different IM30 rings are presented•Helix 7 and loop 2 are located at opposite sides of IM30 rings•IM30-mediated membrane fusion requires a Janus-faced IM30 ring
The IM30 protein is a membrane fusion protein that forms large oligomeric rings from tetrameric building blocks. Saur et al. present 3D reconstructions of the Janus-faced IM30 ring, which illustrates membrane binding properties as well as the membrane fusion activity of IM30.
Hemocyanin transports O
in the hemolymph of many arthropod species. Such respiratory proteins have long been considered unnecessary in Myriapoda. As a result, the presence of hemocyanin in Myriapoda ...has long been overlooked. We analyzed transcriptome and genome sequences from all major myriapod taxa - Chilopoda, Diplopoda, Symphyla, and Pauropoda - with the aim of identifying hemocyanin-like proteins.
We investigated the genomes and transcriptomes of 56 myriapod species and identified 46 novel full-length hemocyanin subunit sequences in 20 species of Chilopoda, Diplopoda, and Symphyla, but not Pauropoda. We found in Cleidogona sp. (Diplopoda, Chordeumatida) a hemocyanin-like sequence with mutated copper-binding centers, which cannot bind O
. An RNA-seq approach showed markedly different hemocyanin mRNA levels from ~ 6 to 25,000 reads per kilobase per million reads. To evaluate the contribution of hemocyanin to O
transport, we specifically studied the hemocyanin of the centipede Scolopendra dehaani. This species harbors two distinct hemocyanin subunits with low expression levels. We showed cooperative O
binding in the S. dehaani hemolymph, indicating that hemocyanin supports O
transport even at low concentration. Further, we demonstrated that hemocyanin is > 1500-fold more highly expressed in the fertilized egg than in the adult.
Hemocyanin was most likely the respiratory protein in the myriapod stem-lineage, but multiple taxa may have independently lost hemocyanin and thus the ability of efficient O
transport. In myriapods, hemocyanin is much more widespread than initially appreciated. Some myriapods express hemocyanin only at low levels, which are, nevertheless, sufficient for O
supply. Notably, also in myriapods, a non-respiratory protein similar to insect storage hexamerins evolved from the hemocyanin.
Due to the current poaching crisis in Africa, increasing numbers of white rhinoceroses (Ceratotherium simum) require opioid immobilisation for medical interventions or management procedures. ...Alarmingly, the results of both blood gas analysis and pulse oximetry regularly indicate severe hypoxaemia. Yet, the recovery of the animals is uneventful. Thus, neither of the techniques seems to represent the real oxygenation level. We hypothesized that unusual haemoglobin characteristics of this species interfere with the techniques developed and calibrated for the use in human patients.
Haemoglobin was isolated from blood samples of four adult, white rhinoceroses. Oxygen dissociation curves at pH 7.2 and 7.4 (37°C) were determined based on the absorbance change of haemoglobin in the Soret-region (around 420 nm). Absorbance spectra of oxy- and deoxyhaemoglobin extending into the infrared region were measured.
Oxygen dissociation curves of rhinoceros haemoglobin showed the typical high oxygen affinity (p50 of 2.75 ± 0.07 and 2.00 ± 0.04 kPa for pH 7.2 and 7.4, respectively) under near-physiological conditions with respect to pH, temperature and DPG. The infrared absorbance spectra of oxy- and deoxyhaemoglobin showed only marginal deviations from standard human spectra, possibly due to the presence of a few percent of methaemoglobin in vitro.
Our data enables the development of a rhinoceros-specific blood gas analysis algorithm, which allows for species-specific calculation of SaO2 levels in anaesthetized animals. The inconspicuous absorbance spectra do not contribute to the systematic underestimation of SpO2 by pulse-oximetry.
The DNA repair protein O
-methylguanine-DNA-methyltransferase (MGMT) is a key determinant of cancer resistance. The MGMT inhibitors O
-benzylguanine (O
BG) and O
-(4-bromothenyl)guanine (O
BTG) ...failed to enhance the therapeutic response due to toxic side effects when applied in combination with alkylating chemotherapeutics, indicating a need of inhibitor targeting. We assessed MGMT targeting that relies on conjugating the inhibitors O
BG and O
BTG to ß-D-glucose, resulting in O
BG-Glu and O
BTG-Glu, respectively. This targeting strategy was selected by taking advantage of high demand of glucose in cancers. Contrary to our expectation, the uptake of O
BG-Glu and O
BTG-Glu was not dependent on glucose transporters. Instead, it seems that after membrane binding the conjugates are taken up via flippases, which normally transport phospholipids. This membrane binding is the consequence of the amphiphilic character of the conjugates, which at higher concentrations lead to the formation of micelle-like particles in aqueous solution. The unusual uptake mechanism of the conjugates highlights the importance of proper linker selection for a successful ligand-based drug delivery strategy. We also demonstrate that proteins of the P4-Type ATPase family are involved in the transport of the glucose conjugates. The findings are not only important for MGMT inhibitor targeting, but also for other amphiphilic drugs.
The pore forming hemolysin A, Hla, is a major virulence factor of
. Apparently, 1-2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ...ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla, complement and other pore forming proteins, presumably by increasing membrane permeability. Indeed, in experiments employing rabbit erythrocytes, inhibitory concentrations of frequently employed P2XR-antagonists were in a similar range as previously reported for erythrocytes of other species and other toxins. However, Hla-dependent hemolysis was not enhanced by extracellular ATP, and oxidized adenosinetriphosphate (oxATP) had only a minor inhibitory effect. Unexpectedly, P2XR-inhibitors also prevented Hla-induced lysis of pure lipid membranes, demonstrating that the inhibition did not even depend on the presence of P2XR. Fluorescence microscopy and gel-electrophoresis clearly revealed that P2XR-inhibitors interfere with binding and subsequent oligomerisation of Hla with membranes. Similar results were obtained employing HaCaT-cells. Furthermore, calorimetric data and hemolysis experiments with Hla pre-treated with pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) showed that this compound directly binds to Hla. Our results call for a critical re-assessment of the appealing concept, which suggests that P2XR are general amplifiers of damage by pore-forming proteins.
Scale formation, the deposition of certain minerals such as CaCO3, MgCO3, and CaSO4·2H2O in industrial facilities and household devices, leads to reduced efficiency or severe damage. Therefore, ...incrustation is a major problem in everyday life. In recent years, double hydrophilic block copolymers (DHBCs) have been the focus of interest in academia with regard to their antiscaling potential. In this work, we synthesized well-defined blocklike PAA-PAMPS copolymers consisting of acrylic acid (AA) and 2-acrylamido-2-methyl-propane sulfonate (AMPS) units in a one-step reaction by RAFT polymerization. The derived copolymers had dispersities of 1.3 and below. The copolymers have then been investigated in detail regarding their impact on the different stages of the crystallization process of CaCO3. Ca2+ complexation, the first step of a precipitation process, and polyelectrolyte stability in aqueous solution have been investigated by potentiometric measurements, isothermal titration calorimetry (ITC), and dynamic light scattering (DLS). A weak Ca2+ induced copolymer aggregation without concomitant precipitation was observed. Nucleation, early particle growth, and colloidal stability have been monitored in situ with DLS. The copolymers retard or even completely suppress nucleation, most probably by complexation of solution aggregates. In addition, they stabilize existing CaCO3 particles in the nanometer regime. In situ AFM was used as a tool to verify the coordination of the copolymer to the calcite (104) crystal surface and to estimate its potential as a growth inhibitor in a supersaturated CaCO3 environment. All investigated copolymers instantly stopped further crystal growth. The carboxylate richest copolymer as the most promising antiscaling candidate proved its enormous potential in scale inhibition as well in an industrial-filming test (Fresenius standard method).
PDF
