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High-resolution model of Arabidopsis Photosystem II reveals the structural consequences of digitonin-extraction
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0003-0864-9798
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0003-0807-0348
Umeå University, Faculty of Science and Technology, Department of Chemistry.ORCID iD: 0000-0002-9492-5113
2021 (English)In: Scientific Reports, E-ISSN 2045-2322, Vol. 11, no 1, article id 15534Article in journal (Refereed) Published
Abstract [en]

In higher plants, the photosynthetic process is performed and regulated by Photosystem II (PSII). Arabidopsis thaliana was the first higher plant with a fully sequenced genome, conferring it the status of a model organism; nonetheless, a high-resolution structure of its Photosystem II is missing. We present the first Cryo-EM high-resolution structure of Arabidopsis PSII supercomplex with average resolution of 2.79 Å, an important model for future PSII studies. The digitonin extracted PSII complexes demonstrate the importance of: the LHG2630-lipid-headgroup in the trimerization of the light-harvesting complex II; the stabilization of the PsbJ subunit and the CP43-loop E by DGD520-lipid; the choice of detergent for the integrity of membrane protein complexes. Furthermore, our data shows at the anticipated Mn4CaO5-site a single metal ion density as a reminiscent early stage of Photosystem II photoactivation.

Place, publisher, year, edition, pages
Nature Publishing Group, 2021. Vol. 11, no 1, article id 15534
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-186557DOI: 10.1038/s41598-021-94914-xISI: 000683319500011Scopus ID: 2-s2.0-85111686355OAI: oai:DiVA.org:umu-186557DiVA, id: diva2:1584358
Available from: 2021-08-11 Created: 2021-08-11 Last updated: 2024-01-30Bibliographically approved
In thesis
1. Light’EM up!: structural characterization of light-driven membrane protein complexes by cryogenic electron microscopy
Open this publication in new window or tab >>Light’EM up!: structural characterization of light-driven membrane protein complexes by cryogenic electron microscopy
2024 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Light’EM up! : strukturell karakterisering av ljusdrivna membranproteinkomplex med kryogen elektronmikroskopi
Abstract [en]

Photosynthesis is probably the most important process for allowing life to develop into the diverse forms we see today. In this process, solar radiation is used to convert CO2 into biomass. From this process, we obtain oxygen to breathe, sources of food (plant biomass), and the potential for clean and sustainable energy. Photosystem II (PSII) – a key enzyme in photosynthesis –, is a protein complex located in the thylakoid membrane of photosynthetic organisms. PSII and its light-harvesting antennae capture light energy, driving a charge separation process, which leads to the extraction of electrons from water molecules, forming and releasing molecular oxygen. A PSII dimer is composed of more than 20 unique proteins and hundreds of cofactors which fine-tune the mechanisms of light-harvesting and water oxidation, and stabilize the whole complex. While the arrangement of most (but not all!) of these proteins and cofactors is known, their dynamics and individual contributions are not yet fully understood.

In my thesis work, I took on the challenge of resolving the structure of large protein complexes, such as PSII complexes from various photosynthetic organisms, using a technique called cryogenic electron microscopy (cryo-EM). This PhD dissertation focuses on structurally describing these macromolecular assemblies and how their components (protein, cofactors, and substrate) interact with each other or with their immediate cellular environment.

Among the several outcomes of my research on PSII, I would like to highlight the following findings: 1) the usage of digitonin as a detergent to solubilize PSII destroys the catalytic activity and changes LHCII pigment content, among other consequences; 2) PSII does not seem to incorporate chlorophyll (Chl) a molecules with a farnesyl tail, and the Chl tails’ flexibility justifies not resolving the full-length of some of these molecules in PSII structures. We concluded that flexibility may be an advantage to PSII function; 3) cryo-EM is a technique with the potential to reveal information about electron/proton transfer processes within PSII, and provided us with data, for instance, to suggest a pathway for the protonation of QB, the final electron acceptor in PSII.

In another project, also using cryo-EM, I studied the structure of the S-layer Deinoxanthin Binding Complex (SDBC), a membrane protein complex from Deinococcus radiodurans. This complex is an essential part of the cell envelope, the outermost barrier of this bacterium, and it is known to bind a carotenoid called deinoxanthin, which has significant spectroscopic and antioxidant properties. Additionally, we studied the function of this complex and showed that the SDBC is a quencher of UVC-UVB radiation and reactive oxygen species, with superoxide dismutase activity. This complex has an α-β coiled-coil stalk long enough to reach the inner membrane of the cell envelope.

In summary, visualizing the structural organization and chemistry within these complexes allowed us to gain a new understanding of their function and diversity. Furthermore, this work demonstrates the potential of cryo-EM as a method to render complementary information at resolution superior to state-of-the-art X-ray diffraction methods.

Abstract [sv]

Fotosyntesen är förmodligen den viktigaste processen för att liv ska kunna utvecklas till de olika former vi ser idag. I denna process används solstrålning för att omvandla CO2 till biomassa. Från denna process får vi syre att andas, källor till mat (växtbiomassa) och potentialen för ren och hållbar energi. Fotosystem II (PSII) - ett nyckelenzym i fotosyntesen - är ett proteinkomplex som finns i thylakoidmembranet hos fotosyntetiserande organismer. PSII och dess antenner fångar upp ljusenergi och driver en laddningsseparationsprocess som leder till att elektroner extraheras från vattenmolekyler, vilket bildar och frigör molekylärt syre. PSII består av mer än 20 unika proteiner och hundratals kofaktorer som finjusterar mekanismerna för ljusinsamling och vattenoxidation, och stabiliserar hela komplexet. Även om de flesta (men inte alla!) av dessa proteiner och kofaktorer är kända, är deras dynamik och individuella bidrag ännu inte klarlagda.

I mitt avhandlingsarbete antog jag utmaningen att lösa strukturen hos stora proteinkomplex, såsom PSII-komplex från olika fotosyntetiska organismer, med hjälp av en teknik som kallas kryogen elektronmikroskopi (cryo-EM). Denna avhandling fokuserar på att strukturellt beskriva dessa makromolekylära sammansättningar och hur deras komponenter (protein, kofaktorer och substrat) interagerar med varandra eller med deras omedelbara cellulära miljö.

Bland de resultat från min forskning om PSII skulle jag vilja lyfta fram följande: 1) Användningen av digitonin som detergent för att solubilisera PSII förstör den katalytiska aktiviteten och förändrar LHCII:s pigmentinnehåll, bland andra konsekvenser. 2) PSII verkar inte införliva klorofyll (Chl) a-molekyler med en farnesylsvans, och Chl-svansarnas flexibilitet motiverar att vissa av dessa molekyler inte har full längd i PSII:s strukturer. Vi drog slutsatsen att flexibilitet kan vara en fördel för PSII:s funktion; 3) cryo-EM är en teknik med potential att avslöja information om elektronöverföringsprocesser inom PSII, och gav oss data, till exempel för att föreslå en väg för protonering av QB, den slutliga elektronacceptorn i PSII.

I ett annat projekt, där jag också använde cryo-EM, studerade jag strukturen hos S-layer Deinoxanthin Binding Complex (SDBC), ett membranproteinkomplex från Deinococcus radiodurans. Detta komplex är en viktig del av cellhöljet, den yttersta barriären hos denna bakterie, och det är känt att det binder en karotenoid som kallas deinoxanthin, som har betydande spektroskopiska och antioxidativa egenskaper. Vi studerade dessutom komplexets funktion och visade att SDBC är en släckare av UVC-UVB-strålning och reaktiva syreföreningar, med superoxiddismutasaktivitet. Komplexet har en α-β-spiralstjälk som är tillräckligt lång för att nå cellhöljets inre membran.

Sammanfattningsvis har visualiseringen av den strukturella organisationen och kemin i dessa komplex gett oss en ny förståelse för deras funktion och mångfald. Dessutom visar detta arbete potentialen hos cryo-EM som en metod för att återge data med kompletterande kvalitet och upplösning som är överlägsen de modernaste röntgendiffraktionsmetoderna.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2024. p. 116
Keywords
3D reconstruction, Arabidopsis thaliana, assembly, chlorophyll, cryo-EM, cyanobacteria, Deinococus radiodurans, electron, land plants, membrane protein complexes, Photosystem II, proton, single-particle analysis, S-Layer Deinoxanthin Binding Complex (SDBC), water.
National Category
Structural Biology Biochemistry and Molecular Biology
Research subject
Biochemistry; biological chemistry
Identifiers
urn:nbn:se:umu:diva-220229 (URN)9789180702782 (ISBN)9789180702775 (ISBN)
Public defence
2024-02-23, Aula Biologica (BIO.E.203), Biologihuset, 09:30 (English)
Opponent
Supervisors
Available from: 2024-02-02 Created: 2024-01-30 Last updated: 2024-02-02Bibliographically approved

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Graça, André T.Hall, MichaelPersson, KarinaSchröder, Wolfgang P.

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