- Email: [email protected]
Membrane protein targeting into mitochondria
Abstracts
3. C. Cheng et al., Ion transport in complex layered graphene-based membranes, Science Advances, (2016). doi:10.1016/j.bbabio.2016.04.265
13.03 Membrane protein targeting into mitochondria German Legkuna,, Ivan Okhrimenkoa, Vladimir Chupina, Valentin Gordeliya,b,c a Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia b Institut de Biologie Structurale J.-P. Ebel, Université Grenoble Alpes-CEACNRS, F-38000 Grenoble, France c Institute of Complex Systems (ICS-6), Research Centre Jülich, 52425 Jülich, Germany E-mail address: [email protected] (G. Legkun) Mitochondrion is an essential part of most eukaryotes. These organelles play a key role in energy production and are involved in many processes like apoptosis and aging.Control of mitochondrial processes is quite challenging and designing of transgenic organisms specifically responding to light is an actual problem of optogenetics [1,2]. Photosensitive proteins, especially rhodopsins, are widely used in optogenetics to control physiological activity of the cells. Here we report a functional expression of light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium in mitochondria. The protein has been heterologously expressed in Pichia pastoris mitochondria which is a useful model organism for different studies due to its short culture time and relative easiness of engineering. Western blot analysis and fast transient absorption spectroscopy proved that gene constructs were expressed and processed to mature protein. Correct localization was also supported by fluorescent microscopy data. References 1. Hara, K. Y., Wada, T., Kino, K., Asahi, T., & Sawamura, N. Construction of photoenergetic mitochondria in cultured mammalian cells. Scientific reports (2013), 3 , DOI: 10.1038/srep01635 2. Hoffmann, A., Hildebrandt, V., Heberle, J., & Büldt, G. Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe. Proceedings of the National Academy of Sciences USA 91(20) (1994), 9367–9371. doi:10.1016/j.bbabio.2016.04.266
13.04 A gold nanoparticle based assay for the detection of inhibitors of bacterial cytochrome bd oxidases Anton Nikolaeva, Hamid Nasirib, Eugenie Fourniera, Jo Hoeserc, Thorsten Friedrichc, Petra Hellwiga, Frederic Melina, a Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, Strasbourg, France b Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Straße 7, D-60438 Frankfurt am Main, Germany c Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Freiburg, Germany E-mail address: [email protected] (F. Melin) Cytochrome bd oxidases are respiratory membrane proteins expressed only in bacteria, including the human pathogens Escherichia coli, Mycobacterium tuberculosis and Klebsiella pneumonia [1,2]. They are
e125
believed to play an important role in the virulence, adaptability and resistance of these bacteria. They thus constitute a potential target for the discovery of novel antibiotics with different modes of action. We describe an electrochemical assay for the study of E. coli cytochrome bd oxidase activity and inhibition with quinone binding site tool compounds. The setup relies on direct electron transfer to/from the heme cofactors of this large enzyme at electrodes specifically modified with gold nanoparticles [3]. After optimization of the protein coverages, the assay shows at pH 7 a good reproducibility, sensitivity and stability over time. Inhibition of the enzyme was probed first with N-oxo-2heptyl-4-Hydroxyquinoline (HQNO). Half maximal inhibitory concentration (IC50) values in the micromolar range were obtained, which is consistent with previous studies [4]. The screening of small molecules derived from quinones is also presented. References 1. S. Jünemann, Cytochrome bd terminal oxidase, Biochim. Biophys. Acta, 1321 (1997) 107–127 2. V.B. Borisov, R.B. Gennis, J. Hemp, M.I. Verkhovsky, The cytochrome bd respiratory oxygen reductases, Biochim. Biophys. Acta, 1807 (2011) 1398–1413 3. T. Meyer, F. Melin, H. Xie, I. von der Hocht, S.K. Choi, M.R. Noor, H. Michel, R.B. Gennis, T. Soulimane, P. Hellwig, Evidence for Distinct Electron Transfer Processes in Terminal Oxidases from Different Origin by Means of Protein Film Voltammetry, J. Am. Chem. Soc., 136 (2014) 10854–10857 4. B. Meunier, S.A. Madgwick, E. Reil, W. Oettmeier, P.R. Rich, New inhibitors of the quinol oxidation sites of bacterial cytochromes bo and bd, Biochemistry, 34 (1995) 1076–1083. doi:10.1016/j.bbabio.2016.04.267
13.05 Imaging and manipulation of temperature in single living cells for thermal biology Kohki Okabea,b,, Takashi Funatsua a Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan b JST, PRESTO, Tokyo, Japan Corresponding author at: Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan E-mail address: [email protected] (K. Okabe) Temperature, a key regulator of biochemical reactions, influences many physiological functions of organisms. Recent progress in intracellular thermometry shows temporal and spatial variation associated with cellular functions, shedding light on an intriguing hypothesis: temperature change inside of a cell is essentially involved in cell functions. Conceiving this idea, we have been investigating intracellular temperature based on a fluorescent polymeric thermometer and quantitative fluorescence imaging. We first performed the tracking of the averaged temperature of a single cell and showed that the averaged temperature of single COS7 cells increased upon mitochondrial uncoupling [1]. Next, we developed a novel method to visualize intracellular temperature distribution by utilizing fluorescence lifetime imaging microscopy (FLIM), which indicated an interesting temperature gradient observed between the nucleus and the cytoplasm at the steady-state and the local temperature change provoked by endogenous heat production from mitochondria in COS7 cells [2]. Furthermore, by introducing a simple and artificial heat source using infra-red (IR) laser irradiation, we have monitored the transient temperature elevation in single living cells to observe intracellular heat
3. C. Cheng et al., Ion transport in complex layered graphene-based membranes, Science Advances, (2016). doi:10.1016/j.bbabio.2016.04.265
13.03 Membrane protein targeting into mitochondria German Legkuna,, Ivan Okhrimenkoa, Vladimir Chupina, Valentin Gordeliya,b,c a Moscow Institute of Physics and Technology, 141700 Dolgoprudny, Moscow Region, Russia b Institut de Biologie Structurale J.-P. Ebel, Université Grenoble Alpes-CEACNRS, F-38000 Grenoble, France c Institute of Complex Systems (ICS-6), Research Centre Jülich, 52425 Jülich, Germany E-mail address: [email protected] (G. Legkun) Mitochondrion is an essential part of most eukaryotes. These organelles play a key role in energy production and are involved in many processes like apoptosis and aging.Control of mitochondrial processes is quite challenging and designing of transgenic organisms specifically responding to light is an actual problem of optogenetics [1,2]. Photosensitive proteins, especially rhodopsins, are widely used in optogenetics to control physiological activity of the cells. Here we report a functional expression of light-driven proton pump bacteriorhodopsin (bR) from Halobacterium salinarium in mitochondria. The protein has been heterologously expressed in Pichia pastoris mitochondria which is a useful model organism for different studies due to its short culture time and relative easiness of engineering. Western blot analysis and fast transient absorption spectroscopy proved that gene constructs were expressed and processed to mature protein. Correct localization was also supported by fluorescent microscopy data. References 1. Hara, K. Y., Wada, T., Kino, K., Asahi, T., & Sawamura, N. Construction of photoenergetic mitochondria in cultured mammalian cells. Scientific reports (2013), 3 , DOI: 10.1038/srep01635 2. Hoffmann, A., Hildebrandt, V., Heberle, J., & Büldt, G. Photoactive mitochondria: in vivo transfer of a light-driven proton pump into the inner mitochondrial membrane of Schizosaccharomyces pombe. Proceedings of the National Academy of Sciences USA 91(20) (1994), 9367–9371. doi:10.1016/j.bbabio.2016.04.266
13.04 A gold nanoparticle based assay for the detection of inhibitors of bacterial cytochrome bd oxidases Anton Nikolaeva, Hamid Nasirib, Eugenie Fourniera, Jo Hoeserc, Thorsten Friedrichc, Petra Hellwiga, Frederic Melina, a Laboratoire de Bioelectrochimie et Spectroscopie, UMR 7140, Chimie de la Matière Complexe, Université de Strasbourg, CNRS, Strasbourg, France b Johann Wolfgang Goethe-University Frankfurt, Max-von-Laue-Straße 7, D-60438 Frankfurt am Main, Germany c Albert-Ludwigs-Universität Freiburg, Institut für Biochemie, Freiburg, Germany E-mail address: [email protected] (F. Melin) Cytochrome bd oxidases are respiratory membrane proteins expressed only in bacteria, including the human pathogens Escherichia coli, Mycobacterium tuberculosis and Klebsiella pneumonia [1,2]. They are
e125
believed to play an important role in the virulence, adaptability and resistance of these bacteria. They thus constitute a potential target for the discovery of novel antibiotics with different modes of action. We describe an electrochemical assay for the study of E. coli cytochrome bd oxidase activity and inhibition with quinone binding site tool compounds. The setup relies on direct electron transfer to/from the heme cofactors of this large enzyme at electrodes specifically modified with gold nanoparticles [3]. After optimization of the protein coverages, the assay shows at pH 7 a good reproducibility, sensitivity and stability over time. Inhibition of the enzyme was probed first with N-oxo-2heptyl-4-Hydroxyquinoline (HQNO). Half maximal inhibitory concentration (IC50) values in the micromolar range were obtained, which is consistent with previous studies [4]. The screening of small molecules derived from quinones is also presented. References 1. S. Jünemann, Cytochrome bd terminal oxidase, Biochim. Biophys. Acta, 1321 (1997) 107–127 2. V.B. Borisov, R.B. Gennis, J. Hemp, M.I. Verkhovsky, The cytochrome bd respiratory oxygen reductases, Biochim. Biophys. Acta, 1807 (2011) 1398–1413 3. T. Meyer, F. Melin, H. Xie, I. von der Hocht, S.K. Choi, M.R. Noor, H. Michel, R.B. Gennis, T. Soulimane, P. Hellwig, Evidence for Distinct Electron Transfer Processes in Terminal Oxidases from Different Origin by Means of Protein Film Voltammetry, J. Am. Chem. Soc., 136 (2014) 10854–10857 4. B. Meunier, S.A. Madgwick, E. Reil, W. Oettmeier, P.R. Rich, New inhibitors of the quinol oxidation sites of bacterial cytochromes bo and bd, Biochemistry, 34 (1995) 1076–1083. doi:10.1016/j.bbabio.2016.04.267
13.05 Imaging and manipulation of temperature in single living cells for thermal biology Kohki Okabea,b,, Takashi Funatsua a Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan b JST, PRESTO, Tokyo, Japan Corresponding author at: Graduate School of Pharmaceutical Sciences, the University of Tokyo, Tokyo, Japan E-mail address: [email protected] (K. Okabe) Temperature, a key regulator of biochemical reactions, influences many physiological functions of organisms. Recent progress in intracellular thermometry shows temporal and spatial variation associated with cellular functions, shedding light on an intriguing hypothesis: temperature change inside of a cell is essentially involved in cell functions. Conceiving this idea, we have been investigating intracellular temperature based on a fluorescent polymeric thermometer and quantitative fluorescence imaging. We first performed the tracking of the averaged temperature of a single cell and showed that the averaged temperature of single COS7 cells increased upon mitochondrial uncoupling [1]. Next, we developed a novel method to visualize intracellular temperature distribution by utilizing fluorescence lifetime imaging microscopy (FLIM), which indicated an interesting temperature gradient observed between the nucleus and the cytoplasm at the steady-state and the local temperature change provoked by endogenous heat production from mitochondria in COS7 cells [2]. Furthermore, by introducing a simple and artificial heat source using infra-red (IR) laser irradiation, we have monitored the transient temperature elevation in single living cells to observe intracellular heat