Periodic Reporting for period 1 - PHOTO-LINK (Harnessing the Sun: Structural elucidation of the photoprotective Photosystem II interactome)
Reporting period: 2022-11-15 to 2024-11-14
Oxygenic photosynthesis, the foundation of nearly all life on Earth, converts water and sunlight into chemical energy, sustaining ecosystems and driving primary energy production. Over 3.5 billion years of evolution, the Photosystem II (PSII) enzyme, responsible for water splitting, has remained fundamentally conserved, while its associated regulatory proteins have evolved to enable photosynthetic organisms to thrive across diverse habitats. These dynamic interactions, crucial for light harvesting and photoprotection, underlie the adaptability of photosynthesis and its vital role in energy and food security. Understanding how PSII and its interactome adapt to changing environments is essential for addressing global challenges like climate change, food demand, and renewable energy production.
The PHOTO-LINK project investigates the structural and functional adaptations of PSII, focusing on Arabidopsis thaliana (At), a model land plant, and Phaeodactylum tricornutum (Pt), a marine diatom. It aims to:
> Achieve comprehensive structural descriptions of Photosystem II components in At and Pt under standard conditions.
> Elucidate the dynamic structural changes within the “fast” and “slow” PSII interactomes in response to varying light conditions.
> Advance integrative structural proteomics methodologies for application in photosynthesis research.
The work’s significance lies in bridging knowledge gaps regarding photosynthetic adaptations and laying a foundation for innovative applications in sustainable agriculture, bioenergy, and environmental research. This aligns with EU priorities such as the Green Deal and Horizon Europe’s sustainability and innovation goals.
The PHOTO-LINK project investigates the structural and functional adaptations of PSII, focusing on Arabidopsis thaliana (At), a model land plant, and Phaeodactylum tricornutum (Pt), a marine diatom. It aims to:
> Achieve comprehensive structural descriptions of Photosystem II components in At and Pt under standard conditions.
> Elucidate the dynamic structural changes within the “fast” and “slow” PSII interactomes in response to varying light conditions.
> Advance integrative structural proteomics methodologies for application in photosynthesis research.
The work’s significance lies in bridging knowledge gaps regarding photosynthetic adaptations and laying a foundation for innovative applications in sustainable agriculture, bioenergy, and environmental research. This aligns with EU priorities such as the Green Deal and Horizon Europe’s sustainability and innovation goals.
Using advanced structural proteomics and cryo-electron microscopy (cryo-EM), The project focused on two key areas: (WP1) establishing comprehensive structural profiles of PSII complexes under standard conditions and (WP2) elucidating dynamic structural changes during photoprotection and repair mechanisms under varying light conditions. The major technical challenge to overcome has been to establish and optimize the structural proteomics workflow, which gather information which and how proteins interacts, in combination with in vivo physiological measurements.
Structural Profiling: Optimized crosslinking methods, leveraging enrichable PhoX and TMPAC to preserve protein-protein interactions and enhance peptide identification in At and Pt. Methods successfully captured transient interactions under varying light conditions.
Main Achievements includes:
> Dynamic Studies: Conducted time-framed crosslinking to analyze light-dependent interactions
> Mapping “fast” photoprotection mechanisms in two plant species.
> Cryo-EM structural characterization of Diatom's PSII complexes under high-light conditions.
> Integration of Tools and Training: Advanced integrative XL-MS workflows for physiological conditions. Established knowledge-sharing collaborations and trained researchers in cutting-edge proteomics techniques.
Structural Profiling: Optimized crosslinking methods, leveraging enrichable PhoX and TMPAC to preserve protein-protein interactions and enhance peptide identification in At and Pt. Methods successfully captured transient interactions under varying light conditions.
Main Achievements includes:
> Dynamic Studies: Conducted time-framed crosslinking to analyze light-dependent interactions
> Mapping “fast” photoprotection mechanisms in two plant species.
> Cryo-EM structural characterization of Diatom's PSII complexes under high-light conditions.
> Integration of Tools and Training: Advanced integrative XL-MS workflows for physiological conditions. Established knowledge-sharing collaborations and trained researchers in cutting-edge proteomics techniques.
WP1.1 [Experimental set-up] & WP1.2 [XL-MS on intact thylakoid membranes and PSIIsc purification]
We introduced an innovative cross-linking approach using the enrichable chemical crosslinker named PhoX, combined with a cationic compound. We showed that this combination significantly improved cross-linking efficiency and the identification of unique cross-linked peptides.
Key Results:
• The combination of PhoX and TMPAC showed minimal effects on photosynthetic activity (as measured by Fv/Fm and ETR), confirming that this methodology can be applied under physiological conditions without disrupting the photosynthetic function of the membranes (Fig.1).
• TMPAC treatment enhanced the number of unique cross-linked peptides by up to 56%, significantly improving the yield compared to control samples (Fig. 2).
• Structural analysis of cross-linked peptides revealed transmembrane interactions in photosystem II, with cross-linking distances typically under 35 Å, supporting the biological relevance of the identified complexes.
WP2.1 [Proteome-wide PSII interactome structural dynamics in land plants during HL treatments]
We optimized the protocol to be able to capture labile interactions within a short time window and a quantitative approach to study the dynamics of protein-protein interaction networks during light acclimation. For this we first screened the optimal experimental setup to be able to crosslink functional thylakoids in a short-time window of 5 minutes. After the reaction is started we quench with NH3 and quickly freeze the thylakoid sample, In parallel, we measured the photo-physiological response. We were able to relatively quantify the light-dependent interactions during light exposure with dozens of novel protein interaction with both PSII and PSI were detected, some of them potentially representing novel regulatory actors regulating light use efficiency.
WP2.2 [Structural characterization of stabilized PSIIsc with interacting regulatory proteins during acclimation to HL conditions in Pt]
We managed to effectively crosslink intact Pt cells. Then, we were able to purify in sufficient amount a quality some PSII and PSI sub-complexes (possibly assembly/disassembly intermediates). We then focused on stabilizing some of these complexes upon light treatment and analyze them by cryo-EM and single particle analysis, as part of the secondment at IBS. The relevant datasets were acquired and some have already produced promising results that are now being analyzed.
__ Overall, the PHOTO-LINK project results were Beyond the State of the Art for:
> Novel Methodologies: PHOTO-LINK has advanced XL-MS and cryo-EM methodologies, enabling the capture of dynamic protein-protein interactions under physiological conditions. These approaches offer new insights into photosynthetic adaptation and regulatory mechanisms.
> Scientific Insights: The project revealed the dynamics of PSII interactomes during light acclimation, identifying novel protein complexes and transient interactions critical to photosynthesis regulation.
> Technological Applications: High-resolution structural maps of PSII and PSI complexes contribute to advancing bioenergy research and improving crop resilience under environmental stress.
__ Key Needs for Further Uptake
Further Research: Build on identified transient protein interactions to refine structural models and validate findings through orthogonal approaches.
Demonstration and Scaling: Pilot the application of PHOTO-LINK methodologies across diverse photosynthetic organisms to assess generalizability and scalability.
We introduced an innovative cross-linking approach using the enrichable chemical crosslinker named PhoX, combined with a cationic compound. We showed that this combination significantly improved cross-linking efficiency and the identification of unique cross-linked peptides.
Key Results:
• The combination of PhoX and TMPAC showed minimal effects on photosynthetic activity (as measured by Fv/Fm and ETR), confirming that this methodology can be applied under physiological conditions without disrupting the photosynthetic function of the membranes (Fig.1).
• TMPAC treatment enhanced the number of unique cross-linked peptides by up to 56%, significantly improving the yield compared to control samples (Fig. 2).
• Structural analysis of cross-linked peptides revealed transmembrane interactions in photosystem II, with cross-linking distances typically under 35 Å, supporting the biological relevance of the identified complexes.
WP2.1 [Proteome-wide PSII interactome structural dynamics in land plants during HL treatments]
We optimized the protocol to be able to capture labile interactions within a short time window and a quantitative approach to study the dynamics of protein-protein interaction networks during light acclimation. For this we first screened the optimal experimental setup to be able to crosslink functional thylakoids in a short-time window of 5 minutes. After the reaction is started we quench with NH3 and quickly freeze the thylakoid sample, In parallel, we measured the photo-physiological response. We were able to relatively quantify the light-dependent interactions during light exposure with dozens of novel protein interaction with both PSII and PSI were detected, some of them potentially representing novel regulatory actors regulating light use efficiency.
WP2.2 [Structural characterization of stabilized PSIIsc with interacting regulatory proteins during acclimation to HL conditions in Pt]
We managed to effectively crosslink intact Pt cells. Then, we were able to purify in sufficient amount a quality some PSII and PSI sub-complexes (possibly assembly/disassembly intermediates). We then focused on stabilizing some of these complexes upon light treatment and analyze them by cryo-EM and single particle analysis, as part of the secondment at IBS. The relevant datasets were acquired and some have already produced promising results that are now being analyzed.
__ Overall, the PHOTO-LINK project results were Beyond the State of the Art for:
> Novel Methodologies: PHOTO-LINK has advanced XL-MS and cryo-EM methodologies, enabling the capture of dynamic protein-protein interactions under physiological conditions. These approaches offer new insights into photosynthetic adaptation and regulatory mechanisms.
> Scientific Insights: The project revealed the dynamics of PSII interactomes during light acclimation, identifying novel protein complexes and transient interactions critical to photosynthesis regulation.
> Technological Applications: High-resolution structural maps of PSII and PSI complexes contribute to advancing bioenergy research and improving crop resilience under environmental stress.
__ Key Needs for Further Uptake
Further Research: Build on identified transient protein interactions to refine structural models and validate findings through orthogonal approaches.
Demonstration and Scaling: Pilot the application of PHOTO-LINK methodologies across diverse photosynthetic organisms to assess generalizability and scalability.