- Email: [email protected]
Taxonomic Distribution and Characterization of the Alternative Oxidase in Animals
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Abstracts
03.23 Taxonomic Distribution and Characterization of the Alternative Oxidase in Animals Allison E. McDonald Department of Biology, Wilfrid Laurier University, Waterloo, Canada E-mail address: [email protected] (A.E. McDonald) Alternative oxidase (AOX) is a ubiquinol oxidase present in the electron transport systems of many organisms that has been best characterized in plants and the protist Trypanosoma brucei. AOX is also present in a wide variety of animals including members of the phyla Placozoa, Porifera, Nematoda, Arthropoda, Mollusca, Annelida, Rotifera, Brachiopoda, Cnidaria, Echinodermata, Hermichordata, and Chordata. Our recent work has focused on taking a comparative and integrated approach to the study of this enzyme in animals using two different experimental systems. The first project involves the use of a heterologous yeast expression system to express the AOX of the Pacific oyster (Crassostrea gigas) in order to examine AOX activity in isolated yeast mitochondria. Oyster AOX is correctly targeted to the mitochondria of the yeast, is active, and displays resistance to cyanide and sensitivity to n-propyl gallate as evaluated using high-resolution respirometry. The second project uses the intertidal marine copepod Tigriopus califonicus as a model system for the study of the role of AOX in environmental stress acclimation and adaptation in a whole animal. Molecular biology techniques have confirmed the presence of AOX DNA and the expression of AOX mRNA is this species and we are currently developing tools to study the AOX protein in this system. The characterization of animal AOXs and an understanding of protein activity regulation in these species must be completed before a proposed use as a gene therapy tool to treat human mitochondrial diseases can be contemplated. doi:10.1016/j.bbabio.2016.04.310
03.24 Isolation and crystallization of the pathological mutants of human dihydrolipoamide dehydrogenase Bálint Nagy, Ágnes Hubert, Eszter Szabó, Zsófia Zámbó, Vera Ádám-Vizi, Attila Ambrus Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary E-mail address: [email protected] (A. Ambrus) The human alpha-ketoglutarate dehydrogenase complex (hKGDHc) has a key role in the energy production of cells by catalyzing an irreversible step in the Krebs cycle; hKGDHc produces succinyl-CoA and NADH from -ketoglutarate, CoA and NAD+. Under pathological conditions hKGDHc is a significant source of reactive oxygen species (ROS) and proposed to have a fundamental role in the pathogeneses of several neurological and cardiological diseases; ROS is generated via the E3 component (dihydrolipoamide dehydrogenase) of hKGDHc. Our laboratory previously has revealed that selected disease-causing mutations of hE3 stimulate the ROS generation by isolated hE3. We seek to investigate the structural and mechanistic bases of this modulated ROS generation using various biophysical and structural approaches including X-ray crystallography. Here, we purified to homogeneity two disease-causing mutants of hE3 (K37E, E340K) using our previously optimized expression and purification protocol for hE3; the proteins were expressed in E. coli BL21(DE3) cells from the pET52b+ plasmid vector and purified by FPLC affinity chromatography
in a single step. The mutants were screened for crystallization using commercially available kits and our optimization solutions. Crystals were yet found for K37E only. To investigate hKGDHc reconstituted with hE3 mutants, we also intended to optimize the heterologous expression in E. coli and the purification of the E1 and E2 components. Besides the high-level expression of E2, we were also able to attain the purification of this component to its homogeneity; expression optimization is currently underway for E1. doi:10.1016/j.bbabio.2016.04.311
03.25 Effect of Hofmeister cosolutes on the photocycle of bacteriorhodopsin Dávid Nagy, András Dér, László Zimányi Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary E-mail address: [email protected] (L. Zimányi) The photocycle of bacteriorhodopsin (bR) was studied by kinetic absorption spectroscopy from 250 ns to seconds, at the sufficiently high pH=7.5 to avoid pH-dependent branching, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit (SVD-EFASM) [1] of the flash-induced difference spectra provided evidence for five spectrally but at least six kinetically distinct intermediates. Both in the absence and in the presence of salts the same overall photocycle characteristics with comparable rate constants and corresponding amplitude spectra were found. Nevertheless, the phenomenological rate constants systematically depended on both the presence of salt and on the type of anion in the medium. The effect of high salt concentrations manifested itself in the deceleration of the last two exponential processes, resulting in a slower recovery of the parent bR state. Hofmeister effect was exclusively observed on the rate of exponential process no. 4, in the ms range. This process corresponds to the main phase of the M to N transition of the photocycle of wild type bR. In the presence of kosmotropic and neutral anions (fluoride, sulfate, acetate, chloride) this rate was the same, while with increasingly chaotropic anions (iodide, perchlorate, thiocyanate) it accelerated by up to 60%. The distinct Hofmeister effect on the bR photocycle will be discussed based on available spectroscopic and X-ray structural data, and on a recent model of the Hofmeister effect on the structural stability of proteins [2]. References 1. L. Zimányi, Analysis of the bacteriorhodopsin photocycle by singular value decomposition with self-modeling: A critical evaluation using realistic simulated data, J. Phys. Chem. B 108 (2004) 4199-4209 2. A. Dér, L. Kelemen, L. Fábián, S. G. Taneva, E. Fodor, T. Páli, A. Cupane, M. G. Cacace, J. J. Ramsden, Interfacial water structure controls protein conformation, J. Phys. Chem. B 111 (2007) 5344-5350. doi:10.1016/j.bbabio.2016.04.312
03.26 Phylogenomic analysis of the type I NADH:quinone-oxidoreductase German E. Novakovskya, Daria V. Dibrovab, Armen Y. Mulkidjaniana,b,c a School of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia
Abstracts
03.23 Taxonomic Distribution and Characterization of the Alternative Oxidase in Animals Allison E. McDonald Department of Biology, Wilfrid Laurier University, Waterloo, Canada E-mail address: [email protected] (A.E. McDonald) Alternative oxidase (AOX) is a ubiquinol oxidase present in the electron transport systems of many organisms that has been best characterized in plants and the protist Trypanosoma brucei. AOX is also present in a wide variety of animals including members of the phyla Placozoa, Porifera, Nematoda, Arthropoda, Mollusca, Annelida, Rotifera, Brachiopoda, Cnidaria, Echinodermata, Hermichordata, and Chordata. Our recent work has focused on taking a comparative and integrated approach to the study of this enzyme in animals using two different experimental systems. The first project involves the use of a heterologous yeast expression system to express the AOX of the Pacific oyster (Crassostrea gigas) in order to examine AOX activity in isolated yeast mitochondria. Oyster AOX is correctly targeted to the mitochondria of the yeast, is active, and displays resistance to cyanide and sensitivity to n-propyl gallate as evaluated using high-resolution respirometry. The second project uses the intertidal marine copepod Tigriopus califonicus as a model system for the study of the role of AOX in environmental stress acclimation and adaptation in a whole animal. Molecular biology techniques have confirmed the presence of AOX DNA and the expression of AOX mRNA is this species and we are currently developing tools to study the AOX protein in this system. The characterization of animal AOXs and an understanding of protein activity regulation in these species must be completed before a proposed use as a gene therapy tool to treat human mitochondrial diseases can be contemplated. doi:10.1016/j.bbabio.2016.04.310
03.24 Isolation and crystallization of the pathological mutants of human dihydrolipoamide dehydrogenase Bálint Nagy, Ágnes Hubert, Eszter Szabó, Zsófia Zámbó, Vera Ádám-Vizi, Attila Ambrus Department of Medical Biochemistry, MTA-SE Laboratory for Neurobiochemistry, Semmelweis University, Budapest, Hungary E-mail address: [email protected] (A. Ambrus) The human alpha-ketoglutarate dehydrogenase complex (hKGDHc) has a key role in the energy production of cells by catalyzing an irreversible step in the Krebs cycle; hKGDHc produces succinyl-CoA and NADH from -ketoglutarate, CoA and NAD+. Under pathological conditions hKGDHc is a significant source of reactive oxygen species (ROS) and proposed to have a fundamental role in the pathogeneses of several neurological and cardiological diseases; ROS is generated via the E3 component (dihydrolipoamide dehydrogenase) of hKGDHc. Our laboratory previously has revealed that selected disease-causing mutations of hE3 stimulate the ROS generation by isolated hE3. We seek to investigate the structural and mechanistic bases of this modulated ROS generation using various biophysical and structural approaches including X-ray crystallography. Here, we purified to homogeneity two disease-causing mutants of hE3 (K37E, E340K) using our previously optimized expression and purification protocol for hE3; the proteins were expressed in E. coli BL21(DE3) cells from the pET52b+ plasmid vector and purified by FPLC affinity chromatography
in a single step. The mutants were screened for crystallization using commercially available kits and our optimization solutions. Crystals were yet found for K37E only. To investigate hKGDHc reconstituted with hE3 mutants, we also intended to optimize the heterologous expression in E. coli and the purification of the E1 and E2 components. Besides the high-level expression of E2, we were also able to attain the purification of this component to its homogeneity; expression optimization is currently underway for E1. doi:10.1016/j.bbabio.2016.04.311
03.25 Effect of Hofmeister cosolutes on the photocycle of bacteriorhodopsin Dávid Nagy, András Dér, László Zimányi Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary E-mail address: [email protected] (L. Zimányi) The photocycle of bacteriorhodopsin (bR) was studied by kinetic absorption spectroscopy from 250 ns to seconds, at the sufficiently high pH=7.5 to avoid pH-dependent branching, in the presence of high concentrations of various salts. Chemometric analysis combined with multiexponential fit (SVD-EFASM) [1] of the flash-induced difference spectra provided evidence for five spectrally but at least six kinetically distinct intermediates. Both in the absence and in the presence of salts the same overall photocycle characteristics with comparable rate constants and corresponding amplitude spectra were found. Nevertheless, the phenomenological rate constants systematically depended on both the presence of salt and on the type of anion in the medium. The effect of high salt concentrations manifested itself in the deceleration of the last two exponential processes, resulting in a slower recovery of the parent bR state. Hofmeister effect was exclusively observed on the rate of exponential process no. 4, in the ms range. This process corresponds to the main phase of the M to N transition of the photocycle of wild type bR. In the presence of kosmotropic and neutral anions (fluoride, sulfate, acetate, chloride) this rate was the same, while with increasingly chaotropic anions (iodide, perchlorate, thiocyanate) it accelerated by up to 60%. The distinct Hofmeister effect on the bR photocycle will be discussed based on available spectroscopic and X-ray structural data, and on a recent model of the Hofmeister effect on the structural stability of proteins [2]. References 1. L. Zimányi, Analysis of the bacteriorhodopsin photocycle by singular value decomposition with self-modeling: A critical evaluation using realistic simulated data, J. Phys. Chem. B 108 (2004) 4199-4209 2. A. Dér, L. Kelemen, L. Fábián, S. G. Taneva, E. Fodor, T. Páli, A. Cupane, M. G. Cacace, J. J. Ramsden, Interfacial water structure controls protein conformation, J. Phys. Chem. B 111 (2007) 5344-5350. doi:10.1016/j.bbabio.2016.04.312
03.26 Phylogenomic analysis of the type I NADH:quinone-oxidoreductase German E. Novakovskya, Daria V. Dibrovab, Armen Y. Mulkidjaniana,b,c a School of Bioengineering and Bioinformatics, Moscow State University, Moscow 119992, Russia