Difference between revisions of "Calisto F"

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== MitoEAGLE Short-Term Scientific Mission ==
 
== MitoEAGLE Short-Term Scientific Mission ==
 +
****: [[Short-Term_Scientific_Missions_MitoEAGLE#STSM_Grant_Period_4 |STSM Grant Period 4]]
 +
::: '''Work Plan summary'''
 +
:::: Electron transfer respiratory chains generate the transmembrane difference of electrochemical potential that is the energy source for ATP synthesis, solute transport and motility. In many organisms respiratory chains differ in composition and organization1. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing essentially the same catalytic reactions. The diversity and apparent redundancy of prokaryotic respiratory chains reflects the versatility and robustness of their organisms. The oxidation of quinol with subsequent reduction of cytochrome c seemed until 1999 to evade the paradigm of diversified respiratory chains in prokaryotes. The reaction was thought to be exclusively catalyzed by the bc1/b6f complex2, also known as complex III. This notion changed with the identification of alternative complex III (ACIII), a new quinol:cytochrome c/HiPIP (High Potential Iron–sulfur Protein) oxidoreductase that was first observed in Rhodothermus marinus3. ACIII is widespread in bacteria1 and mainly present in organisms that lack the bc1/b6f complex. In addition to reducing periplasmatic proteins, such as HiPIP and soluble cytochrome c, ACIII can directly transfer electrons from quinol to the caa3 terminal oxidase without the intervention of any soluble electron carrier4. Besides the functional linkage of ACIII with the caa3 oxygen reductase, the structural association of the two complexes into a supercomplex was also observed in R. marinus4,5. Even though ACIII is functionally equivalent to the cytochrome bc1 complex, these two enzymes are structurally unrelated5. In R. marinus, ACIII is composed of seven subunits encoded by the Act gene cluster4, six of which are conserved across species (ActA to ActF). Recently, in the structural characterization of ACIII, we identified two putative proton pathways and one quinol-binding site5. These results provided the first direct evidence that ACIII operates by a redox-driven proton translocation mechanism, totally unrelated to the Q-cycle of cytochrome bc1 complex.
 +
 +
:::: The main objective of this project is to investigate the energy transduction mechanism of ACIII. Specifically, measure the membrane potential generation and test the proton pump translocation of ACIII in proteoliposomes, using pH-sensitive dyes. Addressing these questions will definitively contribute to the knowledge of energy transduction mechanism of ACIII.
 +
 +
:::: Specific tasks
 +
:::: 1 –ACIII will be reconstituted into liposomes in the absent and present of the pH-sensitive dye pyranine;
 +
:::: 2 – Measurement of membrane potential will be performed with ACIII proteoliposomes in the presence of a fluorescent dye sensitive to the electrical membrane potential, DiSC3(5) (negative inside);
 +
:::: 3 – Proton translocation will be monitored by the pH-sensitive dye pyranine encapsulated in ACIII proteoliposomes;
 +
:::: 4 - Proton pumping will be measured using a micro pH electrode.
 +
:::: The time required to perform all these experiments is estimated to be up to 2 months.
 +
 +
:::: Host laboratory
 +
:::: The work will be performed in the laboratory of Prof. Dr. Christoph von Ballmoos at the Department of Chemistry and Biochemistry, University of Bern (http://vonballmoos.dcb.unibe.ch/). His laboratory is mainly dedicated to the study of the function and molecular mechanism of membrane proteins, applying different biochemical and biophysical techniques, including protein reconstitution into liposomes and measurement of membrane potential and proton translocation with fluorescent dyes and pH electrode. They have experience with a variety of bioenergetic enzymes, including complex I, cytochrome c oxidase, quinol oxidase and ATP synthase and Na+/H+ antiporters. Very recently, they have applied the above-mentioned techniques to characterize the H+/e- stoichiometry of the bo3 oxidase of the bacterium Vitreoscilla6. In this project, we will directly collaborate Simone Graf, the first author of this publication that has conducted all the experiments. Their experience in functional reconstitution of membrane protein into liposomes and subsequent analysis is an added value in this project and should increase its likelihood of success.
 +
 +
 
****: [[Short-Term_Scientific_Missions_MitoEAGLE#STSM_Grant_Period_3 |STSM Grant Period 3]]
 
****: [[Short-Term_Scientific_Missions_MitoEAGLE#STSM_Grant_Period_3 |STSM Grant Period 3]]
 
::: '''Work Plan summary'''
 
::: '''Work Plan summary'''

Latest revision as of 10:35, 30 August 2019


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COST Action CA15203 MitoEAGLE
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Calisto F


MitoPedia topics: EAGLE 

COST: Member


Name Calisto Filipa, PhD student
Institution Biological Energy Transduction Group

ITQB-UNL

Address Av. da República

Estação Agronómica Nacional, 2780-157

City Oeiras
State/Province
Country Portugal
Email fcalisto@itqb.unl.pt
Weblink http://www.itqb.unl.pt/bet
O2k-Network Lab


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MitoEAGLE Short-Term Scientific Mission

Work Plan summary
Electron transfer respiratory chains generate the transmembrane difference of electrochemical potential that is the energy source for ATP synthesis, solute transport and motility. In many organisms respiratory chains differ in composition and organization1. Prokaryotes can use a wide range of electron donors and acceptors and may have alternative complexes performing essentially the same catalytic reactions. The diversity and apparent redundancy of prokaryotic respiratory chains reflects the versatility and robustness of their organisms. The oxidation of quinol with subsequent reduction of cytochrome c seemed until 1999 to evade the paradigm of diversified respiratory chains in prokaryotes. The reaction was thought to be exclusively catalyzed by the bc1/b6f complex2, also known as complex III. This notion changed with the identification of alternative complex III (ACIII), a new quinol:cytochrome c/HiPIP (High Potential Iron–sulfur Protein) oxidoreductase that was first observed in Rhodothermus marinus3. ACIII is widespread in bacteria1 and mainly present in organisms that lack the bc1/b6f complex. In addition to reducing periplasmatic proteins, such as HiPIP and soluble cytochrome c, ACIII can directly transfer electrons from quinol to the caa3 terminal oxidase without the intervention of any soluble electron carrier4. Besides the functional linkage of ACIII with the caa3 oxygen reductase, the structural association of the two complexes into a supercomplex was also observed in R. marinus4,5. Even though ACIII is functionally equivalent to the cytochrome bc1 complex, these two enzymes are structurally unrelated5. In R. marinus, ACIII is composed of seven subunits encoded by the Act gene cluster4, six of which are conserved across species (ActA to ActF). Recently, in the structural characterization of ACIII, we identified two putative proton pathways and one quinol-binding site5. These results provided the first direct evidence that ACIII operates by a redox-driven proton translocation mechanism, totally unrelated to the Q-cycle of cytochrome bc1 complex.
The main objective of this project is to investigate the energy transduction mechanism of ACIII. Specifically, measure the membrane potential generation and test the proton pump translocation of ACIII in proteoliposomes, using pH-sensitive dyes. Addressing these questions will definitively contribute to the knowledge of energy transduction mechanism of ACIII.
Specific tasks
1 –ACIII will be reconstituted into liposomes in the absent and present of the pH-sensitive dye pyranine;
2 – Measurement of membrane potential will be performed with ACIII proteoliposomes in the presence of a fluorescent dye sensitive to the electrical membrane potential, DiSC3(5) (negative inside);
3 – Proton translocation will be monitored by the pH-sensitive dye pyranine encapsulated in ACIII proteoliposomes;
4 - Proton pumping will be measured using a micro pH electrode.
The time required to perform all these experiments is estimated to be up to 2 months.
Host laboratory
The work will be performed in the laboratory of Prof. Dr. Christoph von Ballmoos at the Department of Chemistry and Biochemistry, University of Bern (http://vonballmoos.dcb.unibe.ch/). His laboratory is mainly dedicated to the study of the function and molecular mechanism of membrane proteins, applying different biochemical and biophysical techniques, including protein reconstitution into liposomes and measurement of membrane potential and proton translocation with fluorescent dyes and pH electrode. They have experience with a variety of bioenergetic enzymes, including complex I, cytochrome c oxidase, quinol oxidase and ATP synthase and Na+/H+ antiporters. Very recently, they have applied the above-mentioned techniques to characterize the H+/e- stoichiometry of the bo3 oxidase of the bacterium Vitreoscilla6. In this project, we will directly collaborate Simone Graf, the first author of this publication that has conducted all the experiments. Their experience in functional reconstitution of membrane protein into liposomes and subsequent analysis is an added value in this project and should increase its likelihood of success.


Work Plan summary
I would like to apply to a short-term scientific mission grant, which will allow me to understand the electron transfer pathway and the proton translocation mechanism of Alternative Complex III. I am a PhD student at ITQB in the Biological Energy Transduction group under the supervision of Manuela M. Pereira. I am dedicated to the study of the Alternative Complex III (ACIII), a protein from bacteria respiratory chain, which has the same quinol:cytochrome c oxidoreductase activity as the cytochrome bc1 complex. Recently, I published as first coauthor an article on the structural characterization of ACIII (Nat Commun. 2018 doi: 10.1038/s41467-018-04141-8) where we identified two putative proton pathways and one quinol-binding. These results provide the first direct evidence that ACIII operates by a redox-driven proton translocation mechanism, totally unrelated to the Q-cycle of cytochrome bc1 complex. In order to extend this characterization, I would like to test the proton pump activity of ACIII in liposomes, using fluorescence and advance membrane protein purification/reconstitution methodologies pioneered in Dr Duncan G.G. McMillan Lab at the Department of Biotechnology, Delft University of Technology. Thank you for considering my application. Sincerely, Filipa Calisto


MitoEAGLE Inclusiveness Target Countries - Conference Grant

Scientific report - EBEC2018