Advanced

Painfully Energetic : A tale of two proteins potentially connected

Sperling, Eva LU (2017)
Abstract (Swedish)
Varje cell har ett membran som skapar en gräns mot omvärlden och skyddar
cellen. Men cellen måste alltjämt utbyta viktiga molekyler med omvärlden för att
överleva. Därför finns specialiserade proteiner, som sitter i membranen och utgör
portar i denna barriär. Denna avhandling fokuserar på två membranproteiner, en
protonpump och en jonkanal.
Varje dag i våra liv använder vi hjärnan, muskler och hjärtat. Vart och ett av dessa
organ behöver energi. Energivalutan i kroppen kallas ATP (adenosintrifosfat), en
liten molekyl som deltar i många reaktioner i kroppen. ATP-produktion är mycket
komplicerad och många olika enzymer är involverade. Nästan alla organismer
utnyttjar flera stora proteinkomplex vilka är... (More)
Varje cell har ett membran som skapar en gräns mot omvärlden och skyddar
cellen. Men cellen måste alltjämt utbyta viktiga molekyler med omvärlden för att
överleva. Därför finns specialiserade proteiner, som sitter i membranen och utgör
portar i denna barriär. Denna avhandling fokuserar på två membranproteiner, en
protonpump och en jonkanal.
Varje dag i våra liv använder vi hjärnan, muskler och hjärtat. Vart och ett av dessa
organ behöver energi. Energivalutan i kroppen kallas ATP (adenosintrifosfat), en
liten molekyl som deltar i många reaktioner i kroppen. ATP-produktion är mycket
komplicerad och många olika enzymer är involverade. Nästan alla organismer
utnyttjar flera stora proteinkomplex vilka är organiserade i en respirationsskedja. I
eukaryoter (t.ex. djur och växter) finns de i mitokondriernas membran, de
organeller som brukar kallas cellens kraftverk. Många sjukdomar, däribland
Alzheimers och Parkinsons, utvecklas på grund av defekter i mitokondrierna.
Detta leder till energibrist i cellen. Därför är det viktigt att förstå funktion och
struktur av energiproduktionsenzymen. Det största och första enzymet i
respirationsskedjan heter komplex I. Komplex I består av en del som sitter i
membranet och en annan vattenlöslig del som sticker ut från membranet. Man kan
hitta andra proteiner i naturen som är väldigt lika enskilda delar av komplex I, och
förmodligen har komplex I evolverat från släktingar till dessa mindre proteiner
som tillsammans har bildat ett stort proteinkomplex med nya funktioner. Vårt syfte
med detta projekt var att karaktärisera dessa besläktade proteiner och testa om de
fortfarande har liknande funktioner.
Den andra delen handlar om hur vi kan förnimma/känna av vår omvärld. Det är
livsviktigt att vi kan detektera förändringar i omvärlden, t.ex. temperatur, tryck
etc. Vår kropp genomsyras av nervceller som är kopplade till det centrala
nervsystemet (hjärnan och ryggmärgen). Några nervceller har specialiserade
jonkanaler, som kan aktiveras genom förändringar i temperatur, tryck eller
elektrisk spänning. Dessutom kan olika molekyler binda till jonkanaler och
aktivera dem. När en jonkanal aktiveras skickas en elektrisk impuls till centrala
nervsystemet och vi kan reagera. En av de specialiserade jonkanalerna heter
TRPA1 och utgör temat för den andra delen i avhandligen. TRPA1 aktiveras av
kalla och varma temperaturer och många olika molekyler (t.ex. från stark mat som
wasabi och senap). Många människor lider av kronisk smärta, för vilket det saknas
läkemedel utan biverkningar. Därför är TRPA1 ett attraktiv mål i utvecklingen av
nya läkemedel. Av den orsaken är det viktigt att lösa proteinstrukturen, så att man
förstår hur TRPA1 aktiveras. Vårt syfte med denna del av projektet var att
undersöka och minimera en liten strukturell enheten av TRPA1, som fortfarande
fungerar, för att lättare urskilja vilka strukturförändringar som sker vid aktivering
TRPA1. (Less)
Abstract
NADH:quinone oxidoreductase (Complex I) is the first enzyme of the respiratory chain and is involved in energy conservation generating an electro-chemical gradient across a membrane. The enzyme can be divided into a membrane spanning domain and a hydrophilic domain, which protrudes from the membrane. In the hydrophilic domain electrons from NADH oxidation are transported via a wire of iron-sulfur (Fe-S ) clusters to quinone, which is reduced. While the membrane domain is responsible for proton translocation to maintain the proton motive force, which is important for ATP synthesis. Large protein complexes like complex I have evolved from an assembly of discrete functional building blocks of which there are extant homologs. Two very... (More)
NADH:quinone oxidoreductase (Complex I) is the first enzyme of the respiratory chain and is involved in energy conservation generating an electro-chemical gradient across a membrane. The enzyme can be divided into a membrane spanning domain and a hydrophilic domain, which protrudes from the membrane. In the hydrophilic domain electrons from NADH oxidation are transported via a wire of iron-sulfur (Fe-S ) clusters to quinone, which is reduced. While the membrane domain is responsible for proton translocation to maintain the proton motive force, which is important for ATP synthesis. Large protein complexes like complex I have evolved from an assembly of discrete functional building blocks of which there are extant homologs. Two very different protein families, the Mrpantiporter and membrane bound [NiFe]-hydrogenases contain subunits which are homologous to complex I subunits. Part one of this work aimed to better understand the functional relationship between the related protein subunits of complex I, Mrp-antiporter and [NiFe]-hydrogenases. This knowledge will help us to elucidate the proton translocation pathway in complex I. First we compared the functional differentiation of complex I antiporter-like subunits with transporter subunits of the Hyc and Hyf hydrogenases and the 11-subunit complex I. For that we tested if the different subunits could rescue the growth of two salt sensitive Bacillus subtilis strain, which each lacked one of the two large Mrp-antiporter subunits (MrpA/MrpD). The 11-subunit complex I subunits could restore the growth in a similar manner as the complex I subunits, whereas the hydrogenase subunits could substitute equally well for the two MrpA and MrpD. We confirmed that 11-subunit complex I is a bona fide complex I. and that the hydrogenase subunits have intermediate forms of the antiporter-like subunits. Secondly we examined the functional relationship of the two homologous proteins MrpA from the Mrp-antiporter and NuoL from complex I. We located a stretch of amino acid residues which is conserved only in NuoL and MrpA, but not in the other complex I antiporter-like subunits or in MrpD. These residues were subjected to site directed mutagenesis and any resulting effects were examined in vivo by B. subtilis complementation studies and 23Na-NMR. Only one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux in both subunits, the other mutants were all able to cope with high salt levels.

Ion channels are important for many processes in the cell and critically depend on gradients over membranes to execute their functions. They are involved in the detection of changes in the environment, which is an important survival mechanism for every organism. One of these ion channels is TRPA1, which belongs to the TRP superfamily of non-selective cation channels. TRPA1 can be activated by changes in temperature and voltage, as well as by a wide range of electrophilic and non-electrophilic chemicals. As structural information is limited, the exact activation mechanism is still elusive. The aim of the second part was to study the structural and functional changes of TRPA1 upon activation by temperature and chemical activators. We studied the effect of increased temperature and ligands on the conformation of mosquito TRPA1 (AgTRPA1), using intrinsic tryptophan fluorescence, SRCD and nanoDSF. We showed that the electrophilic ligands tested were quenching the tryptophan flourescence in the same way, suggesting a similar binding mechanism. We propose a putative model how temperature and ligand can activate AgTRPA1.Furthermore, we truncated the C-terminal region of human TRPA1, in an attempt to narrow down the minimal structural and functional unit of hTRPA1. This will facilitate future structural and functional studies of the activation mechanism. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Dr. Drew, David, Department of Biochemistry and Biophysics, Stockholm University, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Complex I , Mrp-antiporter, Membrane bound hydrogenases, TRP ion channels, TRPA1, thermosensation, chemosensation, 23Na-NMR
pages
167 pages
publisher
Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology
defense location
Lecture hall A, Center for chemistry and chemical engineering, Naturvetarvägen 14, Lund
defense date
2017-03-31 09:15
ISBN
978-91-7422-508-2
978-91-7422-507-5
language
English
LU publication?
yes
id
7f4dc35f-ede9-45d8-9d21-1711a8218bcc
date added to LUP
2017-03-07 11:25:01
date last changed
2017-03-11 14:16:48
@phdthesis{7f4dc35f-ede9-45d8-9d21-1711a8218bcc,
  abstract     = {NADH:quinone oxidoreductase (Complex I) is the first enzyme of the respiratory chain and is involved in energy conservation generating an electro-chemical gradient across a membrane. The enzyme can be divided into a membrane spanning domain and a hydrophilic domain, which protrudes from the membrane. In the hydrophilic domain electrons from NADH oxidation are transported via a wire of iron-sulfur (Fe-S ) clusters to quinone, which is reduced. While the membrane domain is responsible for proton translocation to maintain the proton motive force, which is important for ATP synthesis. Large protein complexes like complex I have evolved from an assembly of discrete functional building blocks of which there are extant homologs. Two very different protein families, the Mrpantiporter and membrane bound [NiFe]-hydrogenases contain subunits which are homologous to complex I subunits. Part one of this work aimed to better understand the functional relationship between the related protein subunits of complex I, Mrp-antiporter and [NiFe]-hydrogenases. This knowledge will help us to elucidate the proton translocation pathway in complex I. First we compared the functional differentiation of complex I antiporter-like subunits with transporter subunits of the Hyc and Hyf hydrogenases and the 11-subunit complex I. For that we tested if the different subunits could rescue the growth of two salt sensitive Bacillus subtilis strain, which each lacked one of the two large Mrp-antiporter subunits (MrpA/MrpD). The 11-subunit complex I subunits could restore the growth in a similar manner as the complex I subunits, whereas the hydrogenase subunits could substitute equally well for the two MrpA and MrpD. We confirmed that 11-subunit complex I is a bona fide complex I. and that the hydrogenase subunits have intermediate forms of the antiporter-like subunits. Secondly we examined the functional relationship of the two homologous proteins MrpA from the Mrp-antiporter and NuoL from complex I. We located a stretch of amino acid residues which is conserved only in NuoL and MrpA, but not in the other complex I antiporter-like subunits or in MrpD. These residues were subjected to site directed mutagenesis and any resulting effects were examined in vivo by B. subtilis complementation studies and 23Na-NMR. Only one mutation (M258I/M225I) showed differences in the efficiency of cell growth and sodium efflux in both subunits, the other mutants were all able to cope with high salt levels.<br/><br/>Ion channels are important for many processes in the cell and critically depend on gradients over membranes to execute their functions. They are involved in the detection of changes in the environment, which is an important survival mechanism for every organism. One of these ion channels is TRPA1, which belongs to the TRP superfamily of non-selective cation channels. TRPA1 can be activated by changes in temperature and voltage, as well as by a wide range of electrophilic and non-electrophilic chemicals. As structural information is limited, the exact activation mechanism is still elusive. The aim of the second part was to study the structural and functional changes of TRPA1 upon activation by temperature and chemical activators. We studied the effect of increased temperature and ligands on the conformation of mosquito TRPA1 (AgTRPA1), using intrinsic tryptophan fluorescence, SRCD and nanoDSF. We showed that the electrophilic ligands tested were quenching the tryptophan flourescence in the same way, suggesting a similar binding mechanism. We propose a putative model how temperature and ligand can activate AgTRPA1.Furthermore, we truncated the C-terminal region of human TRPA1, in an attempt to narrow down the minimal structural and functional unit of hTRPA1. This will facilitate future structural and functional studies of the activation mechanism.},
  author       = {Sperling, Eva},
  isbn         = {978-91-7422-508-2},
  keyword      = {Complex I ,Mrp-antiporter,Membrane bound hydrogenases,TRP ion channels,TRPA1,thermosensation,chemosensation,23Na-NMR},
  language     = {eng},
  pages        = {167},
  publisher    = {Lund University, Faculty of Science, Department of Chemistry, Division of Biochemistry and Structural Biology},
  school       = {Lund University},
  title        = {Painfully Energetic : A tale of two proteins potentially connected},
  year         = {2017},
}