Recombinant Morus indica Photosystem II reaction center protein H (psbH)

What is the structural position and function of psbH in the Photosystem II supercomplex?

The psbH protein is a small subunit of the Photosystem II (PSII) core complex. It occupies a position near the reaction center and plays a crucial role in the stability of the PSII core. Within the multiprotein PSII supercomplex structure, psbH contributes to maintaining the proper arrangement of other subunits. Functionally, psbH is involved in electron transport and photoprotection mechanisms, helping to prevent photodamage under high light conditions. The protein's strategic position facilitates its interaction with both the reaction center and peripheral components of the PSII assembly .

What techniques are commonly used for isolating and purifying recombinant psbH protein?

The isolation and purification of recombinant psbH typically involves:

• Gene cloning of the psbH sequence from Morus indica into an appropriate expression vector

• Heterologous expression in a suitable host system (commonly E. coli or yeast)

• Cell lysis under mild conditions to preserve protein structure

• Initial purification through affinity chromatography (utilizing a His-tag or other fusion tag)

• Secondary purification via ion exchange chromatography

• Final polishing step using size exclusion chromatography

For membrane proteins like psbH, solubilization with appropriate detergents (such as n-dodecyl-β-D-maltoside) is crucial during the purification process. The choice of detergent significantly affects the structural integrity and functional properties of the isolated protein .

How does light intensity affect the expression and structural dynamics of psbH in Morus indica?

Light intensity has profound effects on both the expression levels and structural configuration of psbH in Morus indica. Research has shown that plants grown under different light intensities (low, moderate, and high) exhibit significant variations in their PSII supercomplex composition and organization.

At increasing light intensities, a structural remodeling of the PSII antenna system occurs, with a notable reduction in the amount of LHCII M-trimers in the isolated complexes. This is evidenced by decreased levels of associated proteins Lhcb3 and Lhcb6. This remodeling does not occur uniformly throughout the thylakoid membrane, suggesting different acclimation strategies depending on membrane regions and light exposure patterns.

High light exposure induces a decrease in the PSII antenna cross-section in isolated supercomplexes and partial depletion of the entire antenna system in thylakoid membranes. This serves as a protective mechanism to prevent photodamage to the reaction center when light continuously exceeds the energy-processing capacity. These adaptive responses involve psbH and highlight its role in the structural flexibility of PSII complexes under varying environmental conditions .

What methodologies are most effective for studying the interaction between psbH and other PSII subunits in recombinant systems?

The most effective methodologies for studying psbH interactions with other PSII subunits include:

• Cryo-electron microscopy (cryo-EM): This technique has revolutionized structural studies of membrane protein complexes, allowing visualization of PSII supercomplexes at near-atomic resolution. Single particle analysis of cryo-EM data can reveal the precise position of psbH and its contacts with neighboring subunits.

• Cross-linking mass spectrometry: Chemical cross-linking followed by mass spectrometric analysis can identify specific interaction sites between psbH and other PSII subunits.

• Co-immunoprecipitation studies: Using antibodies against psbH or potential interaction partners to pull down protein complexes and identify interacting proteins.

• FRET (Förster Resonance Energy Transfer): When combined with fluorescent labeling of recombinant proteins, FRET can detect and characterize proximity and dynamic interactions between psbH and other subunits.

• Molecular dynamics simulations: Computational approaches can model the dynamic behavior of psbH within the PSII complex and predict interaction interfaces.

These methodologies, particularly when combined in an integrative approach, provide complementary information about the structural and functional relationships between psbH and its interaction partners in the PSII complex .

How does the insertion of recombinant psbH affect the functional antenna size of PSII in reconstitution experiments?

The insertion of recombinant psbH into PSII complexes can significantly influence the functional antenna size (ASII). Research indicates that the specific isoform of associated proteins, particularly Lhcb4, plays a crucial role in determining how psbH affects the ASII.

A notable increase in the Lhcb4.3 isoform occurs in high-light acclimated plants compared to low-light plants, both in isolated supercomplexes and thylakoid membranes. Unlike isoforms Lhcb4.1-2, the Lhcb4.3 isoform has a truncated C-terminus located at the binding interface with Lhcb6 within the supercomplex structure. The incorporation of Lhcb4.3 in the PSII-LHCII supercomplex may play a major role in decreasing its functional antenna size by reducing its affinity to bind additional M-trimers.

In reconstitution experiments, recombinant psbH has been observed to influence this dynamic by affecting the binding stability of antenna proteins. Proper incorporation of psbH supports the stable association of core antenna proteins while allowing for the dynamic regulation of peripheral antenna components in response to environmental cues. This highlights the crucial role of psbH in modulating energy capture efficiency and photoprotection .

What protocols are recommended for analyzing the phosphorylation state of psbH from Morus indica?

For analyzing the phosphorylation state of psbH from Morus indica, the following protocol is recommended:

• Sample preparation:

  • Isolated thylakoids or PSII particles should be solubilized in a buffer containing phosphatase inhibitors

  • Immediate flash-freezing in liquid nitrogen to preserve phosphorylation state

• Analytical techniques:

  • Mass spectrometry-based phosphoproteomics

  • Phospho-specific antibody detection via Western blotting

  • Phos-tag SDS-PAGE for mobility shift analysis

  • 2D gel electrophoresis (IEF/SDS-PAGE) followed by immunodetection

• Quantitative assessment:

  • Stable isotope labeling by amino acids in cell culture (SILAC) for quantitative MS analysis

  • Multiple reaction monitoring (MRM) mass spectrometry for targeted quantification

  • Densitometric analysis of Western blots with phospho-specific antibodies

• Validation strategies:

  • In vitro dephosphorylation assays using lambda phosphatase

  • Site-directed mutagenesis of predicted phosphorylation sites

  • Comparative analysis under different light conditions to identify physiologically relevant phosphorylation events

This comprehensive approach allows for robust identification and quantification of phosphorylation sites on psbH, providing insights into how post-translational modifications regulate PSII function under different environmental conditions .

What experimental design is optimal for evaluating the impact of Morus-derived compounds on PSII efficiency?

An optimal experimental design for evaluating Morus-derived compounds on PSII efficiency would include:

• Preparation of test compounds:

  • Extraction and fractionation of Morus leaf or bark material using gradient aqueous-ethanol solutions

  • Purification of specific compounds (e.g., 1-deoxynojirimycin, sanggenon compounds)

  • Preparation of standardized concentrations for testing

• PSII efficiency measurements:

  • Chlorophyll fluorescence analysis (PAM fluorometry)

  • Oxygen evolution measurements using Clark-type electrodes

  • P700 redox kinetics to assess electron transport beyond PSII

  • Thylakoid membrane potential measurements

• Experimental conditions:

  • Tests under varying light intensities (50-1000 μmol photons m⁻² s⁻¹)

  • Temperature variations to assess stress responses (15-35°C)

  • Control vs. treatment comparisons with multiple biological replicates

  • Time-course studies to distinguish immediate vs. long-term effects

• Controls and validations:

  • Positive controls using known PSII inhibitors (DCMU, atrazine)

  • Negative controls with appropriate vehicles (ethanol, DMSO)

  • Dose-response curves to establish effective concentrations

  • Spectroscopic verification of compound-PSII interactions

• Analysis parameters:

  • Maximum quantum yield of PSII (Fv/Fm)

  • Effective quantum yield (ΦPSII)

  • Non-photochemical quenching (NPQ)

  • Electron transport rate (ETR)

  • Recovery kinetics after high light exposure

This comprehensive design allows for robust evaluation of how specific compounds from Morus affect PSII function, potentially identifying molecules that could enhance photosynthetic efficiency or provide photoprotection .

How does the antioxidant capacity of Morus indica extracts correlate with PSII photoprotection mechanisms?

The correlation between antioxidant capacity of Morus indica extracts and PSII photoprotection involves multiple interconnected mechanisms:

The antioxidant compounds in Morus extracts, particularly flavonoids and phenolic acids, directly neutralize reactive oxygen species that would otherwise damage PSII components. These compounds can intercept singlet oxygen and hydroxyl radicals before they reach vulnerable PSII proteins like D1 and psbH.

Additionally, certain Morus compounds appear to enhance the intrinsic photoprotective mechanisms of PSII, including non-photochemical quenching (NPQ) and state transitions. The compounds may stabilize the PSII-LHCII supercomplex structure during high light stress, maintaining optimal energy distribution and preventing photoinhibition.

Research suggests that the photoprotective effects are most pronounced under fluctuating light conditions, where the plant's natural adaptive mechanisms may not respond quickly enough without the support of these antioxidant compounds .

What structural differences exist between psbH from Morus indica compared to psbH from model photosynthetic organisms?

Structural comparisons of psbH from Morus indica with model photosynthetic organisms reveal several notable differences:

The most significant differences appear in the N-terminal and C-terminal domains, while the transmembrane region remains highly conserved across species. The N-terminal domain of Morus indica psbH contains fewer phosphorylation sites compared to Arabidopsis but more than cyanobacterial psbH.

The C-terminal domain of Morus indica psbH shows moderate conservation with other higher plants but significant divergence from algal and cyanobacterial counterparts. This region is implicated in interactions with other PSII subunits and may reflect adaptations to specific environmental conditions experienced by Morus species.

These structural differences likely influence the protein's regulatory properties, particularly its response to varying light conditions and its involvement in PSII supercomplex assembly and stability .

What are the potential applications of recombinant psbH in improving photosynthetic efficiency in stressed environments?

Recombinant psbH holds significant promise for enhancing photosynthetic efficiency under environmental stress conditions through several potential applications:

• Engineered stress resistance: By introducing modified versions of psbH with enhanced stability under heat, drought, or high light conditions, researchers could develop crops with improved photosynthetic performance under stress. Specific modifications to phosphorylation sites or protein-protein interaction domains could enhance PSII repair cycles and photoprotection.

• Optimized energy distribution: Recombinant psbH variants could be designed to modulate the association of antenna complexes with the PSII core, allowing for more efficient light harvesting under low light while preventing overexcitation under high light. This dynamic control of the functional antenna size could significantly improve photosynthetic efficiency across variable light environments.

• Enhanced D1 repair cycle: Since psbH interacts with the D1 protein and influences its turnover during the PSII repair cycle, engineered psbH could potentially accelerate this process under stress conditions, reducing photoinhibition and maintaining higher photosynthetic rates.

• Cross-species optimization: Transferring unique features of psbH from stress-tolerant species like Morus indica to sensitive crop species could potentially confer improved stress tolerance. The remarkable sequence conservation of certain psbH domains, even in distant phylogenetic photosynthetic organisms, suggests that such transfers might be functionally viable.

• Biosensor development: Recombinant psbH fused with reporter proteins could serve as biosensors for monitoring photosynthetic performance in real-time, allowing for rapid assessment of plant responses to changing environmental conditions .

How might advanced structural analysis techniques further elucidate the role of psbH in PSII assembly and repair?

Advanced structural analysis techniques offer promising avenues for deeper understanding of psbH's role in PSII assembly and repair:

• Cryo-electron tomography: This technique could reveal the spatial arrangement of psbH during different stages of PSII assembly and repair in near-native conditions, providing insights into its dynamic interactions with other subunits during these processes.

• Time-resolved X-ray crystallography: By capturing structural snapshots at different stages of PSII assembly and repair, researchers could map the precise conformational changes in psbH and its interaction partners during these processes.

• Single-molecule FRET spectroscopy: This approach would allow direct observation of dynamic structural changes in psbH under different conditions, providing insights into how it responds to environmental cues and contributes to PSII flexibility.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique could identify regions of psbH that undergo conformational changes during assembly, repair, or in response to stress, revealing dynamic structural elements that are not visible in static structures.

• Cross-linking mass spectrometry with targeted mutations: By introducing specific mutations in psbH and analyzing their effects on cross-linking patterns, researchers could map critical interaction interfaces and functional domains with unprecedented precision.

• In situ structural analysis: Techniques like correlative light and electron microscopy could visualize psbH in its native membrane environment, providing context for how its structural dynamics relate to thylakoid membrane organization and PSII megacomplex formation.

What are the current limitations in expressing and purifying functional recombinant psbH protein?

The expression and purification of functional recombinant psbH protein faces several significant challenges:

• Membrane protein expression barriers: As an integral membrane protein, psbH is difficult to express in conventional systems due to potential toxicity to host cells, improper membrane insertion, and protein aggregation. Specialized expression systems like cell-free systems or specific E. coli strains (C41, C43) may be required.

• Maintaining structural integrity: The small size of psbH (approximately 7-8 kDa) and its hydrophobic nature make it particularly challenging to maintain in a properly folded state outside its native environment. The protein often requires specific lipids or detergents to maintain its structure during purification.

• Co-factor requirements: The functional integration of psbH into PSII requires proper association with cofactors and other protein subunits. Reconstituting these interactions in vitro presents significant technical challenges.

• Post-translational modifications: Native psbH undergoes phosphorylation and potentially other modifications that affect its function. Recombinant expression systems may not reproduce these modifications correctly, resulting in functionally distinct protein.

• Verification of functionality: Assessing whether recombinant psbH is functionally equivalent to the native protein requires complex biochemical and biophysical assays, particularly considering its role requires integration into the multi-subunit PSII complex.

• Low expression yields: The combination of these factors typically results in low yields of properly folded, functional protein, making it difficult to obtain sufficient quantities for comprehensive structural and functional studies.

Researchers are addressing these challenges through the development of specialized membrane protein expression systems, novel detergent and lipid nanodisc approaches for membrane protein stabilization, and advanced analytical techniques for characterizing small membrane proteins .

How can researchers optimize experimental conditions to study the phosphorylation dynamics of psbH in Morus indica?

To optimize experimental conditions for studying psbH phosphorylation dynamics in Morus indica, researchers should implement the following strategies:

• Rapid sample preparation protocol:

  • Harvest leaf tissue directly into liquid nitrogen

  • Perform extraction in phosphatase inhibitor-containing buffers (NaF, β-glycerophosphate, and vanadate)

  • Maintain all solutions at 4°C or below throughout the extraction process

  • Complete thylakoid isolation within 30-45 minutes to minimize dephosphorylation

• Light condition standardization:

  • Acclimate plants to specific light intensities (low: 50-100, moderate: 200-300, high: 800-1000 μmol photons m⁻² s⁻¹)

  • Apply controlled light treatments (duration, intensity, spectrum) immediately before harvest

  • Include dark adaptation controls (1-2 hours) to establish baseline phosphorylation

• Advanced detection methods:

  • Develop Morus indica psbH-specific phospho-antibodies

  • Employ Phos-tag™ SDS-PAGE for improved separation of phosphorylated isoforms

  • Utilize targeted MS/MS methods optimized for membrane phosphopeptides

  • Implement parallel reaction monitoring (PRM) for precise quantification

• Time-course experiments:

  • Establish kinetics of phosphorylation/dephosphorylation using short time intervals (30 seconds to 60 minutes)

  • Monitor recovery dynamics following high light exposure

  • Track diurnal patterns of phosphorylation over 24-hour cycles

• Comparative analysis approaches:

  • Compare phosphorylation patterns between different Morus species

  • Correlate phosphorylation states with functional parameters (quantum yield, NPQ)

  • Examine phosphorylation under combined stresses (high light + drought, high light + temperature)

• Validation strategies:

  • Perform in vitro phosphorylation assays with isolated thylakoid membranes

  • Use specific kinase inhibitors to identify responsible kinases

  • Analyze phosphorylation mutants (if available) or create artificial phosphomimetic variants

These optimized conditions would enable researchers to accurately characterize the dynamic phosphorylation states of psbH in Morus indica and correlate these modifications with functional changes in photosynthetic performance under varying environmental conditions .

How might single-molecule techniques advance our understanding of psbH dynamics within the PSII complex?

Single-molecule techniques offer unprecedented opportunities to unravel the dynamic behavior of psbH within the PSII complex:

What computational approaches are most promising for predicting psbH interactions and functional properties?

Several computational approaches show significant promise for predicting psbH interactions and functional properties:

• Molecular dynamics simulations: All-atom and coarse-grained MD simulations can model the dynamic behavior of psbH within the PSII complex embedded in a lipid bilayer. These simulations can predict conformational changes, lipid-protein interactions, and the effects of post-translational modifications on protein dynamics. Advanced techniques like enhanced sampling methods can access longer timescales relevant to PSII assembly and repair.

• Protein-protein docking algorithms: Specialized docking tools for membrane proteins can predict binding interfaces between psbH and other PSII subunits, helping to identify key residues involved in complex assembly and stability. These predictions can guide experimental mutagenesis studies.

• Quantum mechanics/molecular mechanics (QM/MM) methods: These hybrid approaches can model the electronic properties of psbH and its environment, providing insights into how it might influence electron transfer within PSII or participate in redox reactions.

• Machine learning approaches:

  • Deep learning models trained on protein structure databases can predict the impact of mutations on psbH structure and function

  • Graph neural networks can analyze the network of interactions within PSII to identify critical nodes and connections

  • Natural language processing of scientific literature can extract patterns and relationships not obvious from manual review

• Evolutionary coupling analysis: By analyzing patterns of co-evolution in psbH sequences across species, researchers can identify residue pairs that have evolved together, suggesting functional or structural relationships that constrain evolution.

• Multiscale modeling: Integrating simulations across different scales (quantum, molecular, mesoscale) can connect atomic-level details of psbH structure with larger-scale phenomena like thylakoid membrane organization and photosynthetic efficiency.

These computational approaches, particularly when integrated with experimental data, can provide mechanistic insights into psbH function and guide the design of experiments to test specific hypotheses about its role in PSII dynamics .