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Characterization of folding dynamics and accelerated electron transfer in proteins
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology. Linköping University, The Institute of Technology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The research presented in this thesis is divided in two: The first part is a study of the intracellular protein human copper zinc superoxide dismutase which has been associated with the fatale disease Amyotrophic lateral Selerosis (ALS). ALS is a progressive neurodegenerative disease of motor neurons, and in the cases where the disease is inheriled (fALS), roughly 15% is caused by mutations in human copper·zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 causes ALS is still unknown, but it is believed that mutant CuZnSOD proteins misfold followed by aggregation into high molecular species that ultimately lead to the death of motor neurons. In our study we have investigated the dynamical and structural differences between the wt of CuZnSOD and the ALS-associated variant G93A. 1H-15N·HSQC NMR spectroscopy was used to analyze hydrogen deuterium exchange at the backbone amide groups. The study showed that the mutation selectively destabilizes the remote metal binding region. This suggests that the metal binding region might be involved in intermolecular protein-protein interactions which may constitule the eariy stages in formation of aggregates. In another of our studies the monomer to dimer equilibrium as weil as the catalytic activity is investigated upon protein denaturation using CdmCI. The study showed persistent dimer interactions and high catalytic activity at GdmCllevels where the holo-protein according to CD measurements is fully unfolded.

The second part of this thesis focuses on electron transfer (ET) in proteins. ET processes are fundamental in many biological processes such as respiration and photosynthesis. Biological ET reactions occur rapidly over large molecular distances (>20Å) and only minor structural changes around the active site arises during the ET event. Previous work on Rumodified P. aeruginosa azurin have demonstrated that optimized electron coupling through a θ-strand yield a distance decay constant of 1.1 Å-1. ET in biological systems often requires sub-millisecond charge transport over long molecular distances (>20A). This is not possible via direct tunneling through a θ-strand. It is believed that ET rates can be greatly enhanced by multistep tunneling ("hopping") in which redox-active amino acid side-chains act as intermediate donors or acceptors. In our work, rapid spectroscopic methods are used to investigate hopping through an intermediate tryptophan or tyrosine radical. Cu(l) to Re(II) electron tunneling in Re(H107) azurin occurs in the microsecond range, which is much faster than for previously studied Cu(l) to Ru(III) tunneling in Ru(H107). At first it was believed to be multistep tunneling, but further investigation disproved this. A more likely explanalion is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radieal), followed by electron tunneling from Cu(l) to the hole in the imidazole Iigand. In the other investigated system Cu(l) oxidation by a photoexcited Re(l) diimine at position 124 on a ß-strand (His124-Gly123-Trp122-Met121) takes place in nanoseconds, which is remarkable and more than two orders of magnitude faster than for single-step ET at a 19 Adonoracceptor distance. This system is the first model system to show that an intervening tryptophan residue between donor and acceptor can accelerate the ET rate. Therefore this work was published in Science.

Abstract [sv]

l denna avhandling presenteras studier på två olika proteiner med viktiga funktioner i den levande cellen. Proteiner är en mycket viktig grupp av makromolekyler som bland annat ombesörjer energiförsörjning, respiration, nedbrytning av slaggprodukter, transport av näringsämnen och andra makromolekyler, nedbrytning av virus och av bakterier med mera. Ett protein består av aminosyror som är sammanlänkade i en lång kedja i en bestämd sekvens. l naturliga proteiner förekommer 20 olika aminosyror med delvis olika kemiska egenskaper. l de allra flesta fallen så är proteinets funktion och tredimensionella struktur helt bestämd av egenskaperna hos dessa aminosyror och den sekvens de har i aminosyrakedjan. De två projekt som här presenteras är typiska exempel på hur viktig den precisa ordningen i aminosyrakedjan är för att proteiner ska få sin rätta struktur och behålla sin funktion i cellen.

En studie i denna avhandling har fokus på proteinet koppar,zink-superoxiddismutas (CuZnSOD) som är uppbyggt av två likadana proteinenheter (kallad subenheter) som vidhäftar till varandra. Varje proteinenhet innehåller en koppar- och en zinkjon som är viktiga för proteinets funktion. Om endast en aminosyra i den 153 aminosyror !ånga sekvensen byts ut mot en annan sorts amiansyra så kan det orsaka förändringar i proteinet som leder till den dödliga sjukdomen Amyotroph lateral seleros (ALS). Mer än ett hundra olika sådana utbyten av aminosyror på olika platser i proteinet har kopplats till sjukdomen. En relativt väl underbyggd hypotes är att sjukdomen utvecklas på grund av att det sker strukturella förändringar i proteinet som leder till proteinmolekylerna klumpar ihop sig i aggregat som kan skada cellen. l mina studier har jag med en kombination av gentekniska, biokemiska och biofysikaliska metoder undersökt några av de proteinvarianter som orsakar ALS. l en serie experiment har jag mätt hur rörligheten eller stabiliteten i olika delar av proteinet förändrats i de olika varianterna. Genom att mäta hur snabbt vattenmolekyler i proteinets omgivning kan "tränga in i " proteinet och byta en egen vätejon med en bunden vätejon från proteinet kan man beskriva proteinets stabilitet. Resultaten pekar på att aminosyrautbyten på en plats kan ha sin huvudsakliga effekt på en avlägsen plats i samma proteinmolekyl och mera bestämt att den del av proteinet som binder metallerna tycks bli kraftigt påverkad. Resultaten från andra experiment visar att även gränsområdet mellan de två proteindelarna i vissa fall kan påverkas.

Det andra projektet berör elektrontransport inom proteiner. En stor grupp av proteiner i cellen har viktiga funktioner för överföring av energi och transport av elektroner är mycket ofta kopplat till dessa processer. Elektrontransport (ET)över korta avstånd kan ske effektivt via bindningar i proteiner, men vid långa transporter > 20 Å så går den processen för långsamt. l många proteiner måste dock elektronen färdas lång väg och man tror då att den snabbt kan "hoppa" mellan de aromatiska aminosyrorna tryptafan eller tyrosin. Proteinet azurin från Pseudomonas aeruginosa använts som ett modellsystem för studier av ET-reaktioner. l en uppmärksammad studie (publicerad i Science) har vi använt "protein engineering" för att konstruera en snabb väg för elektrontransport och våra resultat visar att en tryptafan som satts in på en plats längs elektronens väg i proteinet accelererade elektrontransporten 1 000-faldigt.

Place, publisher, year, edition, pages
Linköping: Linköpings univeristet , 2008. , 88 p.
Series
Linköping Studies in Science and Technology. Dissertations, ISSN 0345-7524 ; 1218
National Category
Natural Sciences
Identifiers
URN: urn:nbn:se:liu:diva-43776Local ID: 74768ISBN: 978-91-7393-777-1 (print)OAI: oai:DiVA.org:liu-43776DiVA: diva2:264636
Public defence
2008-10-10, Planck, Fysikhuset, Campus Valla, Linköpings universitet, Linköping, 13:00 (English)
Available from: 2009-10-10 Created: 2009-10-10 Last updated: 2012-11-15
List of papers
1. Selective destabilization of the metal binding region caused by the FALS associated mutation G93A in CuZnSOD
Open this publication in new window or tab >>Selective destabilization of the metal binding region caused by the FALS associated mutation G93A in CuZnSOD
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have, by use of 1H-15N-HSQC NMR spectroscopy, analyzed hydrogen exchange at the amide groups of wtCuZnSOD and the FALS-associated G93A SOD-variant in their fully metallated states. From measurements at near physiological conditions we could analyze the exchange at 64% of all backbone amide groups, which have allowed a detailed characterization of the local dynamics at these positions in both the wt and G93A proteins. The results show that the G93A mutation had no effect on the dynamics at a majority of the investigated positions. However the mutation results in local destabilization at the site of mutation and to stabilization at positions that were apparently scattered over the entire protein surface. Most remarkably, the mutation selectively destabilized the remote metal binding region. The results indicate that the metal binding region may be involved in intermolecular protein-protein interactions, which may constitute the early stages in formation of aggregates.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-85309 (URN)
Available from: 2012-11-15 Created: 2012-11-15 Last updated: 2016-05-04
2. The equilibrium between the monomer and dimer form of ALS-related SOD1 mutants
Open this publication in new window or tab >>The equilibrium between the monomer and dimer form of ALS-related SOD1 mutants
(English)Manuscript (preprint) (Other academic)
Abstract [en]

No abstract available.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-85310 (URN)
Available from: 2012-11-15 Created: 2012-11-15 Last updated: 2016-05-04
3. Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins
Open this publication in new window or tab >>Electron tunneling in rhenium-modified Pseudomonas aeruginosa azurins
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2004 (English)In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1655, no 1-3, 59-63 p.Article in journal (Refereed) Published
Abstract [en]

Laser flash-quench methods have been used to generate tyrosine and tryptophan radicals in structurally characterized rhenium-modified Pseudomonas aeruginosa azurins. Cu(I) to “Re(II)” electron tunneling in Re(H107) azurin occurs in the microsecond range. This reaction is much faster than that studied previously for Cu(I) to Ru(III) tunneling in Ru(H107) azurin, suggesting that a multistep (“hopping”) mechanism might be involved. Although a Y108 radical can be generated by flash-quenching a Re(H107)M(II) (M=Cu, Zn) protein, the evidence suggests that it is not an active intermediate in the enhanced Cu(I) oxidation. Rather, the likely explanation is rapid conversion of Re(II)(H107) to deprotonated Re(I)(H107 radical), followed by electron tunneling from Cu(I) to the hole in the imidazole ligand.

Keyword
Electron tunneling, Rhenium complex, Amino acid radical, Blue copper, Azurin
National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-85311 (URN)10.1016/j.bbabio.2003.06.010 (DOI)
Available from: 2012-11-15 Created: 2012-11-15 Last updated: 2017-12-07
4. Tryptophan-accelerated electron flow through proteins
Open this publication in new window or tab >>Tryptophan-accelerated electron flow through proteins
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2008 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 320, no 5884, 1760-1762 p.Article in journal (Refereed) Published
Abstract [en]

Energy flow in biological structures often requires submillisecond charge transport over long molecular distances. Kinetics modeling suggests that charge-transfer rates can be greatly enhanced by multistep electron tunneling in which redox-active amino acid side chains act as intermediate donors or acceptors. We report transient optical and infrared spectroscopic experiments that quantify the extent to which an intervening tryptophan residue can facilitate electron transfer between distant metal redox centers in a mutant Pseudomonas aeruginosa azurin. CuI oxidation by a photoexcited ReI-diimine at position 124 on a histidine(124)-glycine(123)-tryptophan(122)-methionine(121) β strand occurs in a few nanoseconds, fully two orders of magnitude faster than documented for single-step electron tunneling at a 19 angstrom donor-acceptor distance.

National Category
Engineering and Technology
Identifiers
urn:nbn:se:liu:diva-85312 (URN)10.1126/science.1158241 (DOI)
Available from: 2012-11-15 Created: 2012-11-15 Last updated: 2017-12-07

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Museth, Anna Katrine

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