The Membrane-Spanning Domain of Complex I Investigated with Fusion Protein Techniques
(2010)- Abstract
- NADH:quinone oxidoreductase, or complex I, is a large, complex and poorly understood bioenergetic enzyme in the respiratory chain of living organisms. The enzyme has a conserved core structure, comprising fourteen protein subunits: Seven subunits protrude from the membrane and contain a flavin prosthetic group and eight iron-sulfur clusters that guide electrons from the oxidation of NADH towards the quinone binding site(s). The remaining seven subunits make up the membrane domain of the enzyme complex. These proteins are lacking prosthetic groups and thus have no color or other spectral features, and their hydrophobic nature imposes a plethora of other technical obstacles. Nevertheless, it is important to learn more about this part of... (More)
- NADH:quinone oxidoreductase, or complex I, is a large, complex and poorly understood bioenergetic enzyme in the respiratory chain of living organisms. The enzyme has a conserved core structure, comprising fourteen protein subunits: Seven subunits protrude from the membrane and contain a flavin prosthetic group and eight iron-sulfur clusters that guide electrons from the oxidation of NADH towards the quinone binding site(s). The remaining seven subunits make up the membrane domain of the enzyme complex. These proteins are lacking prosthetic groups and thus have no color or other spectral features, and their hydrophobic nature imposes a plethora of other technical obstacles. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery.
In this work a novel type of fusion protein was created, to facilitate studies of the membrane-spanning domain of complex I. The added fusion domain comprises a cytochrome c where an N-terminal membrane anchor helix has been removed to be replaced by a transmembrane segment in the protein to be tagged. The fusion proteins could be quantified with high accuracy using the extinction coefficient for the cytochrome c, but the red color could also be followed with the naked eye. The heme prosthetic group in cytochrome c is covalently bound to the polypeptide, and thus the fusion proteins could also be monitored under denaturing conditions such as after SDS-PAGE. In case of the three large antiporter-like protein subunits NuoL, NuoM and NuoN, unprecedented amounts of the individual proteins could be produced in Escherichia coli, suggesting that the cytochrome domain also protected the proteins from proteolysis. To further improve purification efficiency, a c-terminal his-tag was added, allowing a one step purification process for the individually expressed proteins. Since complex I is notoriously unstable, it is extremely useful to be able to readily monitor the whereabouts of both the soluble domain and the membrane-spanning domain during purification and handling. Therefore, the gene encoding the fused cytochrome c domain was introduced into the nuo-operon in the E. coli chromosome, enabling the production of cytochrome-tagged whole complex I enzyme.
The only limit to the cytochrome c-tagging method is that the c-terminal end of the fusion protein must be periplasmic for heme insertion to occur. A subsequent spin-off from the previous studies was that the transmembrane topology of the small NuoA subunit had to be revised. This revision was also supported by conventional alkaline phosphatase fusion protein techniques. (Less) - Abstract (Swedish)
- Popular Abstract in Swedish
När vi äter kolhydrater, fett, protein och andra näringsämnen sker kemiska reaktioner i kroppen som kan liknas vid dom som sker när man eldar en brasa. Vid förbränning reagerar kolhydrater med syre och bildar koldioxid och vatten. Denna förbränning frigör den energi som funnits lagrad i kolhydraterna, i brasan i form av värme, och i kroppens celler omvandlas den till kemisk energi. Energi förbrukas inte utan omvandlas bara till en annan form. För att ta tillvara den kemiska energin tillverkar kroppen en energibärande molekyl, ATP, som lagrar energin tills den behövs, t ex till att gå och springa.
ATP molekylen tillverkas av en rad stora proteinkomplex som sitter i... (More) - Popular Abstract in Swedish
När vi äter kolhydrater, fett, protein och andra näringsämnen sker kemiska reaktioner i kroppen som kan liknas vid dom som sker när man eldar en brasa. Vid förbränning reagerar kolhydrater med syre och bildar koldioxid och vatten. Denna förbränning frigör den energi som funnits lagrad i kolhydraterna, i brasan i form av värme, och i kroppens celler omvandlas den till kemisk energi. Energi förbrukas inte utan omvandlas bara till en annan form. För att ta tillvara den kemiska energin tillverkar kroppen en energibärande molekyl, ATP, som lagrar energin tills den behövs, t ex till att gå och springa.
ATP molekylen tillverkas av en rad stora proteinkomplex som sitter i cellmembranet i alla celler; bakterier, djur- och människoceller. Dessa komplex samarbetar i något som kallas andningskedjan. Andningskedjan består av fem komplex där dom första fyra samlar tillräckligt med energi för att det femte och sista ska kunna skapa ATP molekylen.
Att förstå hur denna process fungerar på molekylär nivå är mycket viktigt då flera allvarliga sjukdomar kan uppstå om cellen inte kan tillverka tillräckligt med ATP. Några av dessa sjukdomar är Parkinsons, Alzheimers, ALS och Hungtington’s sjukdom (danssjukan).
När man vill studera funktionen av dessa proteinkomplex använder man ofta bakterier. Dessa är enkla att odla i labbmiljö och enkla att genetisk modifiera. När man studerar protein på molekylär nivå utnyttjar man ofta mätbara egenskaper hos proteinet. T ex har tre av dom fyra första komplexen i andningskedjan en så kallad heme-grupp. När proteinet binder en heme-molekyl till sig blir det starkt rödfärgat. Heme-molekylen är den molekyl som gör vårt blod rött.
I det första proteinkomplexet i andningskedjan, Komplex I, finns inga sådana enkelt mätbara grupper som heme-gruppen och detta har bidragit till att man vet väldigt lite om hur just detta proteinkomplexet fungerar.
I våra experiment med att försöka ta reda på mer om Komplex I:s funktion har vi förändrat några av dom olika proteinerna i Komplex I på genetisk väg så att de också kunnat binda till sig en heme grupp och blivit vackert röda. Vi gjorde det genom att använda ett annat protein som innehåller en heme-grupp, cytokrome c. När generna som kodar för de två proteinerna limmas ihop bildas ett fungerande protein med dom två ursprungliga proteinernas egenskaper, ett så kallat fusionsprotein. I vårt fall gjorde fusionen med cytokrom c att även Komplex I blev rött och fick en mätbar egenskap.
Med en hjälp av den röda färgen har vi sedan kunnat rena upp stora mängder av några av dom proteiner som bygger upp Komplex I. Dessa protein kan man nu använda för många olika nya strukturella och funktionella studier av både de enstaka proteindelarna och hela Komplex I enzymet. Den kunskap vi kan skaffa oss framöver kommer hjälpa oss att förstå Komplex I:s funktion i cellen och i andningskedjan, kunskap som är viktig för att t ex kunna ta fram effektiva mediciner mot en rad olika sjukdomar (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1585314
- author
- Trane, Maria LU
- supervisor
- opponent
-
- de Gier, Jan-Willem, Department of Biochemistry and Biophysics, Stockholm University
- organization
- publishing date
- 2010
- type
- Thesis
- publication status
- published
- subject
- keywords
- NuoN, NuoM, NuoL, covalent bound heme, Bacillus subtilis, Escherichia coli, membrane protein, His-tag, Cytochrome c, NADH:quinone oxidureductase, fusion protein
- defense location
- Kemicentrum, Getingevägen 60, Hörsal B
- defense date
- 2010-05-07 10:30:00
- ISBN
- 978-91-7422-240-1
- language
- English
- LU publication?
- yes
- id
- b232ad2f-9cc9-4ab7-99a6-ce217418b778 (old id 1585314)
- date added to LUP
- 2016-04-04 14:28:37
- date last changed
- 2018-11-21 21:20:33
@phdthesis{b232ad2f-9cc9-4ab7-99a6-ce217418b778,
abstract = {{NADH:quinone oxidoreductase, or complex I, is a large, complex and poorly understood bioenergetic enzyme in the respiratory chain of living organisms. The enzyme has a conserved core structure, comprising fourteen protein subunits: Seven subunits protrude from the membrane and contain a flavin prosthetic group and eight iron-sulfur clusters that guide electrons from the oxidation of NADH towards the quinone binding site(s). The remaining seven subunits make up the membrane domain of the enzyme complex. These proteins are lacking prosthetic groups and thus have no color or other spectral features, and their hydrophobic nature imposes a plethora of other technical obstacles. Nevertheless, it is important to learn more about this part of complex I, since it must harbor important parts of the energy coupling machinery. <br/><br>
In this work a novel type of fusion protein was created, to facilitate studies of the membrane-spanning domain of complex I. The added fusion domain comprises a cytochrome c where an N-terminal membrane anchor helix has been removed to be replaced by a transmembrane segment in the protein to be tagged. The fusion proteins could be quantified with high accuracy using the extinction coefficient for the cytochrome c, but the red color could also be followed with the naked eye. The heme prosthetic group in cytochrome c is covalently bound to the polypeptide, and thus the fusion proteins could also be monitored under denaturing conditions such as after SDS-PAGE. In case of the three large antiporter-like protein subunits NuoL, NuoM and NuoN, unprecedented amounts of the individual proteins could be produced in Escherichia coli, suggesting that the cytochrome domain also protected the proteins from proteolysis. To further improve purification efficiency, a c-terminal his-tag was added, allowing a one step purification process for the individually expressed proteins. Since complex I is notoriously unstable, it is extremely useful to be able to readily monitor the whereabouts of both the soluble domain and the membrane-spanning domain during purification and handling. Therefore, the gene encoding the fused cytochrome c domain was introduced into the nuo-operon in the E. coli chromosome, enabling the production of cytochrome-tagged whole complex I enzyme. <br/><br>
The only limit to the cytochrome c-tagging method is that the c-terminal end of the fusion protein must be periplasmic for heme insertion to occur. A subsequent spin-off from the previous studies was that the transmembrane topology of the small NuoA subunit had to be revised. This revision was also supported by conventional alkaline phosphatase fusion protein techniques.}},
author = {{Trane, Maria}},
isbn = {{978-91-7422-240-1}},
keywords = {{NuoN; NuoM; NuoL; covalent bound heme; Bacillus subtilis; Escherichia coli; membrane protein; His-tag; Cytochrome c; NADH:quinone oxidureductase; fusion protein}},
language = {{eng}},
school = {{Lund University}},
title = {{The Membrane-Spanning Domain of Complex I Investigated with Fusion Protein Techniques}},
year = {{2010}},
}