The Glove-like Structure of the Conserved Membrane Protein TatC Provides Insight into Signal Sequence Recognition in Twin-Arginine Translocation

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Highlights? Crystal structures of TatC from the thermophile Aquifex aeolicus are presented ? The architecture of TatC generates a glove-shaped pocket buried in the bilayer ? Molecular dynamics reveal destabilization of the membrane around the pocket ? Correlation to biochemical results suggest the signal sequence binds in this pocket     

Characterization of an anion-exchange porous polypropylene hollow fiber membrane for immobilization of ABL lipase

Hollow fiber membrane offers the advantage to integrate catalytic conversion, product separation and catalyst recovery into a single separation process compared to conventional systems. Polypropylene (PP) hollow fiber membrane is a chemically inert and stable membrane with high potential for enzyme immobilization. The surface properties of polypropylene have been modified by radiation induced graft polymerization. Samples were prepared by grafting of glycidylmethacrylate (GMA) using gamma radiation, at different monomer concentrations and irradiation dose. The resulting epoxy was converted into a diethylamino group as an anion-exchange medium to bind the lipase molecules. Surface properties of the grafted and amine treated samples were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM) and contact angle measurements. AFM revealed higher surface roughness for grafted samples than that of virgin polymer. SEM micrographs illustrated that the porous network was retained at high degree of grafting. Contact angle measurements showed excellent wetting properties with water for the grafted and amine treated membranes. Thermal properties were studied using differential scanning calorimeter (DSC) and thermogravimetic analysis (TGA). It was observed that grafting occurred mainly in the amorphous region of the membranes. Activity and operational stability of ABL lipase, isolated from Arthobacter sp. were assayed after immobilizing it to the modified PP hollow fiber. Immobilized lipase retained 20U/g activity after ten hydrolysis cycles and 68% residual activity after 12 weeks of storage.     

Predicted structures of agonist and antagonist bound complexes of adenosine A3 receptor

We used the GEnSeMBLE Monte Carlo method to predict ensemble of the 20 best packings (helix rotations and tilts) based on the neutral total energy (E) from a vast number (10 trillion) of potential packings for each of the four subtypes of the adenosine G protein-coupled receptors (GPCRs), which are involved in many cytoprotective functions. We then used the DarwinDock Monte Carlo methods to predict the binding pose for the human A(3) adenosine receptor (hAA(3)R) for subtype selective agonists and antagonists. We found that all four A(3) agonists stabilize the 15th lowest conformation of apo-hAA(3)R while also binding strongly to the 1st and 3rd. In contrast the four A(3) antagonists stabilize the 2nd or 3rd lowest conformation. These results show that different ligands can stabilize different GPCR conformations, which will likely affect function, complicating the design of functionally unique ligands. Interestingly all agonists lead to a trans χ1 angle for W6.48 that experiments on other GPCRs associate with G-protein activation while all 20 apo-AA(3)R conformations have a W6.48 gauche+ χ1 angle associated experimentally with inactive GPCRs for other systems. Thus docking calculations have identified critical ligand-GPCR structures involved with activation. We found that the predicted binding site for selective agonist Cl-IB-MECA to the predicted structure of hAA(3)R shows favorable interactions to three subtype variable residues, I253(6.58), V169(EL2), and Q167(EL2), while the predicted structure for hAA(2A)R shows weakened to the corresponding amino acids: T256(6.58), E169(EL2), and L167(EL2), explaining the observed subtype selectivity.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

Phospholamban C-terminal Residues Are Critical Determinants of the Structure and Function of the Calcium ATPase Regulatory Complex

To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val⁴⁹-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex.     

Glycine supports in vivo reduction of nitrate in barley leaves

Glycine, a photorespiratory intermediate, enhanced the in vivo reduction of nitrate in barley (Hordeum vulgare L.) leaf slices, when included in the assay medium. Isonicotinyl hydrazide, an inhibitor of glycine oxidation, partially reduced NO₂⁻ production. The enhancement caused by glycine treatment was reversed by isonicotinyl hydrazide when both were present together in the medium. Similar effects were observed when the excised leaves were preincubated with the metabolite and the inhibitor. Glycine also partially relieved the inhibition of nitrate reduction caused by malonate, an inhibitor of the tricarboxylic acid cycle. The results support the hypothesis that glycine decarboxylation activity is a source of NADH for nitrate reductase activity.