A D-pathway mutation decouples the Paracoccus denitrificans cytochrome c oxidase by altering the side-chain orientation of a distant conserved glutamate

Asparagine 131, located near the cytoplasmic entrance of the D-pathway in subunit I of the Paracoccus denitrificans aa(3) cytochrome c oxidase, is a residue crucial for proton pumping. When replaced by an aspartate, the mutant enzyme is completely decoupled: while retaining full cytochrome c oxidation activity, it does not pump protons. The same phenotype is observed for two other substitutions at this position (N131E and N131C), whereas a conservative replacement by glutamine affects both activities of the enzyme. The N131D variant oxidase was crystallized and its structure was solved to 2.32-A resolution, revealing no significant overall change in the protein structure when compared with the wild type (WT), except for an alternative orientation of the E278 side chain in addition to its WT conformation. Moreover, remarkable differences in the crystallographically resolved chain of water molecules in the D-pathway are found for the variant: four water molecules that are observed in the water chain between N131 and E278 in the WT structure are not visible in the variant, indicating a higher mobility of these water molecules. Electrochemically induced Fourier transform infrared difference spectra of decoupled mutants confirm that the protonation state of E278 is unaltered by these mutations but indicate a distinct perturbation in the hydrogen-bonding environment of this residue. Furthermore, they suggest that the carboxylate side chain of the N131D mutant is deprotonated. These findings are discussed in terms of their mechanistic implications for proton routing through the D-pathway of cytochrome c oxidase.     

Specific heteromeric association of four transmembrane peptides derived from platelet glycoprotein Ib-IX complex

As the receptor on the platelet surface for von Willebrand factor, glycoprotein (GP) Ib-IX complex is critically involved in hemostasis and thrombosis. How the complex is assembled from GP Ibα, GP Ibβ and GP IX subunits, all of which are type I transmembrane proteins, is not entirely clear. Genetic and mutational analyses have identified the transmembrane (TM) domains of these subunits as active participants in assembly of the complex. In this study, peptides containing the transmembrane domain of each subunit have been produced and their interaction with one another characterized. Only the Ibβ TM sequence, but not the Ibα and IX counterparts, can form homo-oligomers in SDS-PAGE and TOXCAT assays. Following up on our earlier observation that a Ibβ-Ibα-Ibβ peptide complex (αβ₂) linked through native juxtamembrane disulfide bonds could be produced from isolated Ibα and Ibβ TM peptides in detergent micelles, we show here that addition of the IX TM peptide facilitates formation of the native αβ₂ complex, reproducing the same effect by the IX subunit in cells expressing the GP Ib-IX complex. Specific fluorescence resonance energy transfer was observed between donor-labeled αβ₂ peptide complex and acceptor-conjugated IX TM peptide in micelles. Finally, the mutation D135K in the IX TM peptide could hamper both the formation of the αβ₂ complex and the energy transfer, consistent with its reported effect in the full-length complex. Overall, our results have demonstrated directly the native-like heteromeric interaction among the isolated Ibα, Ibβ and IX TM peptides, which provides support for the four-helix bundle model of the TM domains in the GP Ib-IX complex and paves the way for further structural analysis. The methods developed in this study may be applicable to other studies of heteromeric interaction among multiple TM helices.     

Rapid incorporation of functional rhodopsin into nanoscale apolipoprotein bound bilayer (NABB) particles

Human apolipoprotein A-I (apo A-I) and its engineered constructs form discoidal lipid bilayers upon interaction with lipids in vitro. We now report the cloning, expression, and purification of apo A-I derived from zebrafish (Danio rerio), which combines with phospholipids to form similar discoidal bilayers and may prove to be superior to human apo A-I constructs for rapid reconstitution of seven-transmembrane helix receptors into nanoscale apolipoprotein bound bilayers (NABBs). We characterized NABBs by gel-filtration chromatography, native polyacrylamide gradient gel electrophoresis, UV-visible photobleaching difference spectroscopy, and fluorescence spectroscopy. We used electron microscopy to determine the stoichiometry and orientation of rhodopsin (rho)-containing NABBs prepared under various conditions and correlated stability and signaling efficiency of rho in NABBs with either one or two receptors. We discovered that the specific activity of G protein coupling for single rhos sequestered in individual NABBs was nearly identical with that of two rhos per NABB under conditions where stoichiometry and orientation could be inferred by electron microscopy imaging. Thermal stability of rho in NABBs was superior to that of rho in various commonly used detergents. We conclude that the NABB system using engineered zebrafish apo A-I is a native-like membrane mimetic system for G-protein-coupled receptors and discuss strategies for rapid incorporation of expressed membrane proteins into NABBs.     

Domain movement of iron sulfur protein in cytochrome bc1 complex is facilitated by the electron transfer from cytochrome b(L) to b(H)

The key step of the "protonmotive Q-cycle" mechanism for cytochrome bc1 complex is the bifurcated oxidation of ubiquinol at the Qp site. ISP is reduced when its head domain is at the b-position and subsequent move to the c1 position, to reduce cytochrome c1, upon protein conformational changes caused by the electron transfer from cytochrome b(L) to b(H). Results of analyses of the inhibitory efficacy and the binding affinity, determined by isothermal titration calorimetry, of Pm and Pf, on different redox states of cytochrome bc1 complexes, confirm this speculation. Pm inhibitor has a higher affinity and better efficacy with the cytochrome b(H) reduced complex and Pf binds better and has a higher efficacy with the ISP reduced complex.     

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II

Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.     

Variations of D (-)-beta-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during post-natal development

1. D(-)-beta-hydroxybutyrate dehydrogenase specific activity of rat liver mitochondria changes during ontogenesis: at birth, the activity is low, then increases to a maximum at 12 days, decreases until 50 days to keep constant thereafter. At the same time, mitochondrial protein amount increases regularly while succinatecytochrome c reductase specific activity slightly increases after birth to keep constant afterwards. 2. The observed changes in activity of D(-)-beta-hydroxybutyrate dehydrogenase are not related to possible interactions between the enzyme and phospholids since addition of lecithin to mitochondria does not change the activity. 3. Electrophoresis of mitochondrial proteins isolated from rats at different development stages demonstrates the presence of a protein band characterized by the same electrophoretic mobility as beta-hydroxybutyrate dehydrogenase and by significative changes of its proportion during maturation: the relative amount of this protein increases from the new-born to the 10-12 days old rat, to decrease afterwards. 4. These findings may signify that the increased activity of the enzyme with a maximum at 10-12 days followed by a decrease is related to the rate of the enzymes biosynthesis.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Phosphorylase activity in relation to starch synthesis in developing Hordeum distichum grain

Activity, control and primer requirements of starch phosphorylase in developing barley endosperm were investigated. Phosphorylase was detected in endosperm extracts from 3 days after anthesis. Unprimed activity was predominant between 2 and 10 days after anthesis, when it constituted 70–80% of total activity, but this proportion declined rapidly as the grain developed. The existence of at least 2 isoenzymes was indicated by studies of pH dependence and phosphate inhibition, and was further supported by acrylamide gel electrophoresis and column chromatography using DEAE-cellulose. The two isoenzymes which ere possibly both glyco proteins, appear in barley endosperm soon after anthesis. One appears capable of unprimed activity, and may be associated with the initiation of a-1,2 glucans, which then serve as primers for starch synthetase. This disappears by 13–15 days after anthesis. The other isoenzyme is capable of some unprimed activity but undergoes modification between 15 and 20 days after anthesis, resulting in the loss of unprimed activity. The relevance of the results to initiation of starch synthesis and to starch synthetase in amyloplasts is discussed.     

Albumin stabilizes 14,15-leukotriene A4

14,15-Leukotriene A4 is a pivotal biosynthetic intermediate in 15-lipoxygenase initiated leukotriene biosynthesis. This compound hydrolyzes instantaneously in phosphate buffer at pH 7.4. However, addition of human or bovine albumin to otherwise identical buffer solutions increases its stability. Intact 14,15-leukotriene A4 then decomposes by first-order kinetics with rate constants inversely proportional to the albumin concentration. Stabilization of 14,15-leukotriene A4 under certain conditions may influence its proportionate transformation by enzymatic vs non-enzymatic processes.     

Evaluation of the anti-inflammatory action of curcumin analog (DM1): Effect on iNOS and COX-2 gene expression and autophagy pathways

This work describes the anti-inflammatory effect of the curcumin-analog compound, sodium 4-[5-(4-hydroxy-3-methoxyphenyl)-3-oxo-penta-1,4-dienyl]-2-methoxy-phenolate (DM1), and shows that DM1 modulates iNOS and COX-2 gene expression in cultured RAW 264.7 cells and induces autophagy on human melanoma cell line A375.