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Complex I in colour - Cytochrome c tagging of complex I

Sperling, Eva (2013) MOBT18 20112
Degree Projects in Molecular Biology
Abstract
Abstract

NADH:ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain. The membrane part contains no redox pigments or prosthetic groups to visualize it in optical spectroscopy like it was done before with the other complexes in the respiratory chain. This fact makes it difficult to detect and quantify the enzyme. Previous experiments in our lab used cytochrome c to tag single subunits of complex I and make them spectroscopically visible.
In this work we tried to obtain a whole complex I that is fused to cytochrome c encoded in the chromosome. For this purpose a truncated version of nuoN was C-terminally fused to cytochrome c by genetic engineering into the gene replacement vector pKOV. The... (More)
Abstract

NADH:ubiquinone oxidoreductase (complex I) is the first and largest enzyme in the respiratory chain. The membrane part contains no redox pigments or prosthetic groups to visualize it in optical spectroscopy like it was done before with the other complexes in the respiratory chain. This fact makes it difficult to detect and quantify the enzyme. Previous experiments in our lab used cytochrome c to tag single subunits of complex I and make them spectroscopically visible.
In this work we tried to obtain a whole complex I that is fused to cytochrome c encoded in the chromosome. For this purpose a truncated version of nuoN was C-terminally fused to cytochrome c by genetic engineering into the gene replacement vector pKOV. The principle is based on homologous recombination and selection with different markers. However the hitherto approaches proved to be unsuccessful, the project of tagging whole complex I with cyt c remains in progress. (Less)
Abstract
Popular science summary

Complex I, let me see your true color

Every day of our lives our body performs many different tasks to let us survive, such as brain activity, muscle contractions or smaller tasks like cell- repair or -division. All those tasks require energy. The energy currency of our body is called ATP (adenosintriphosphate), a molecule that lets us perform many reactions in our body. The production of ATP is rather complicated and involves many different enzymes. Those enzymes sit in eukaryotes (le.g. humans, plants) in a membrane that is situated in the “powerhouses” of our body, the mitochondria.
Scientists discovered that many severe diseases are related to a malfuction in the mitochondria, which can lead to a less... (More)
Popular science summary

Complex I, let me see your true color

Every day of our lives our body performs many different tasks to let us survive, such as brain activity, muscle contractions or smaller tasks like cell- repair or -division. All those tasks require energy. The energy currency of our body is called ATP (adenosintriphosphate), a molecule that lets us perform many reactions in our body. The production of ATP is rather complicated and involves many different enzymes. Those enzymes sit in eukaryotes (le.g. humans, plants) in a membrane that is situated in the “powerhouses” of our body, the mitochondria.
Scientists discovered that many severe diseases are related to a malfuction in the mitochondria, which can lead to a less efficient energy production. That is why it is important to have a closer look on the enzymes, which are responsible for energy production and study their function and structure. Some of those enzymes have a strong red color, because they contain heme groups. Those heme groups help scientists to study the function of those enzymes and to understand their mechanism.
Unfortunately one of the enzymes, Complex I (NADH:ubiquinone oxidoreductase), is colourless, which is one reason why the function of this enzyme is still poorly understood. In this work we tried to give this enyzme a red color to study its function.
Therefore we genetically modified one of the subunits of complex I and added a heme group, so it will be red. We tried then to add the gene of the red subunit into the bacterial chromosome, so that every complex I protein will have a color.

Advisor: Cecilia Hägerhäll
Master´s Degree Project 60 credits, in Microbiology
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Sperling, Eva
supervisor
organization
course
MOBT18 20112
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
4113562
date added to LUP
2013-10-21 10:15:37
date last changed
2013-10-21 10:15:37
@misc{4113562,
  abstract     = {Popular science summary

Complex I, let me see your true color

Every day of our lives our body performs many different tasks to let us survive, such as brain activity, muscle contractions or smaller tasks like cell- repair or -division. All those tasks require energy. The energy currency of our body is called ATP (adenosintriphosphate), a molecule that lets us perform many reactions in our body. The production of ATP is rather complicated and involves many different enzymes. Those enzymes sit in eukaryotes (le.g. humans, plants) in a membrane that is situated in the “powerhouses” of our body, the mitochondria. 
Scientists discovered that many severe diseases are related to a malfuction in the mitochondria, which can lead to a less efficient energy production. That is why it is important to have a closer look on the enzymes, which are responsible for energy production and study their function and structure. Some of those enzymes have a strong red color, because they contain heme groups. Those heme groups help scientists to study the function of those enzymes and to understand their mechanism. 
Unfortunately one of the enzymes, Complex I (NADH:ubiquinone oxidoreductase), is colourless, which is one reason why the function of this enzyme is still poorly understood. In this work we tried to give this enyzme a red color to study its function. 
Therefore we genetically modified one of the subunits of complex I and added a heme group, so it will be red. We tried then to add the gene of the red subunit into the bacterial chromosome, so that every complex I protein will have a color. 

Advisor: Cecilia Hägerhäll
Master´s Degree Project 60 credits, in Microbiology
Department of Biology, Lund University},
  author       = {Sperling, Eva},
  language     = {eng},
  note         = {Student Paper},
  title        = {Complex I in colour - Cytochrome c tagging of complex I},
  year         = {2013},
}