Recombinant Spinacia oleracea Thylakoid lumenal 25.3 kDa protein
Product Specs
Target Background
Q&A
What is the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein and how does it compare to similar proteins in other species?
The Spinacia oleracea Thylakoid lumenal 25.3 kDa protein is a nuclear-encoded protein that is transported to the thylakoid lumen via a bipartite N-terminal peptide sequence. Like other lumenal proteins, it is synthesized in the cytosol, imported into the chloroplast, and then translocated across the thylakoid membrane where its transit peptide is cleaved after translocation. Homologous proteins have been identified in Arabidopsis thaliana and other photosynthetic organisms, suggesting evolutionary conservation of function. In Arabidopsis, several thylakoid lumenal proteins of similar molecular weight have been characterized, with the thylakoid proteome consisting of approximately 100 proteins, about 60 of which have been experimentally verified .
How is the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein categorized within the lumenal proteome?
Based on current classification systems of lumenal proteins, the 25.3 kDa protein would likely be categorized according to its association with the thylakoid membrane:
| Classification | Characteristics | Common Functions | Examples from Studies |
|---|---|---|---|
| Free Lumen (FL) | Soluble proteins freely moving within the lumen | PSII assembly and repair | FKBP16-1, TLP proteins |
| Membrane-Associated Lumen (MAL) | Proteins associated with the inner thylakoid membrane | Support of the oxygen-evolving complex | PsbO, PsbP, PsbQ |
Experimental studies would be needed to determine whether the 25.3 kDa protein functions as a free lumenal protein or a membrane-associated lumenal protein. This classification is functionally significant as FL proteins are typically involved in photosystem assembly and repair, while MAL proteins often support core photosynthetic functions like the oxygen-evolving complex .
What expression systems are most effective for producing recombinant Spinacia oleracea Thylakoid lumenal 25.3 kDa protein?
For the expression of recombinant thylakoid lumenal proteins, multiple systems have been employed with varying success:
| Expression System | Advantages | Challenges | Optimization Strategies |
|---|---|---|---|
| E. coli | High yield, cost-effective, rapid growth | Lack of post-translational modifications, improper folding | Using specialized strains (BL21, Rosetta), lower induction temperature (16-20°C), inclusion of chaperones |
| Yeast (P. pastoris) | Eukaryotic PTMs, proper folding | Lower yield than E. coli | Optimizing methanol induction, using selective media |
| Plant-based systems | Native-like modifications | Lower yield, time-consuming | Transient expression in N. benthamiana leaves |
The methodology for successful expression typically involves cloning the mature protein sequence (without the transit peptide) into an expression vector with an appropriate affinity tag (His6, GST, etc.). Induction conditions must be optimized for each system, with lower temperatures often improving solubility of plant proteins expressed in bacterial systems .
What purification strategies address the challenges of obtaining active recombinant thylakoid lumenal proteins?
Purification of recombinant thylakoid lumenal proteins presents unique challenges due to their specialized folding environments in native conditions. A methodological approach includes:
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Initial capture: Affinity chromatography (IMAC for His-tagged proteins, GST-affinity for GST-fusion proteins)
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Intermediate purification: Ion exchange chromatography to separate based on charge differences
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Polishing step: Size exclusion chromatography for high purity and to assess oligomeric state
For successful refolding of inclusion bodies (if necessary):
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Gradual dilution method using a buffer system containing redox components (e.g., reduced/oxidized glutathione at 1:10 ratio)
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Step-wise dialysis against decreasing concentrations of denaturants
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Monitoring folding by circular dichroism spectroscopy
Activity assays should be performed at each purification stage to ensure the recombinant protein maintains its functional properties. For thylakoid lumenal proteins, this may include assessment of interactions with photosystem components or enzymatic activities specific to the protein .
What structural features of the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein are essential for its function?
While specific structural information for the 25.3 kDa protein needs further investigation, thylakoid lumenal proteins typically possess several key structural features:
| Structural Element | Functional Significance | Research Techniques for Analysis |
|---|---|---|
| N-terminal processing site | Determines correct localization after import | N-terminal sequencing, mass spectrometry |
| Disulfide bonds | Structural stability in oxidizing lumen environment | Non-reducing SDS-PAGE, mass spectrometry |
| Protein-protein interaction domains | Mediation of interactions with photosystem components | Yeast two-hybrid, co-immunoprecipitation |
| Post-translational modification sites | Regulation of activity and turnover | Mass spectrometry, phosphoproteomic analysis |
For thylakoid lumenal proteins, correct folding is particularly important as the lumen provides a unique environment with regulated pH changes during photosynthesis. Proteins in this compartment often form oligomers or participate in dynamic interactions with membrane-embedded photosystem components, which would need to be characterized through techniques like gel filtration chromatography or blue native PAGE .
How does the dynamic nature of the thylakoid lumen affect the Spinacia oleracea 25.3 kDa protein function?
The thylakoid lumen undergoes significant changes in response to photosynthetic activity:
| Condition | Lumen Characteristics | Potential Effects on Protein Function |
|---|---|---|
| Dark | Expanded lumen, neutral pH (~7.5) | Higher protein mobility, potentially different interaction partners |
| Light | Contracted lumen, acidic pH (~5.5-6.0) | Restricted diffusion, altered protein conformation, modified activity |
These dynamic changes likely influence the function of the 25.3 kDa protein through:
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Conformational changes in response to pH fluctuations
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Altered mobility and diffusion rates affecting interaction kinetics
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Modified interaction strengths with binding partners
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Changes in enzymatic activity if the protein possesses catalytic functions
Methodologically, these effects can be studied using fluorescence recovery after photobleaching (FRAP) to measure protein mobility, pH-dependent activity assays, and structural studies under varying pH conditions that mimic the light/dark transitions in the thylakoid lumen .
What are the most effective isolation methods for native Spinacia oleracea Thylakoid lumenal 25.3 kDa protein for comparative studies?
For isolation of native thylakoid lumenal proteins, a step-wise fractionation approach is most effective:
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Chloroplast isolation from fresh spinach leaves using differential centrifugation
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Thylakoid membrane purification by osmotic shock of chloroplasts
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Lumen fraction separation by:
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Mechanical disruption (French pressure cell or sonication) to release free lumenal (FL) proteins
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Washing of disrupted thylakoids to remove remaining FL proteins
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Treatment with 2.6 M urea and 200 mM NaCl to release membrane-associated lumenal (MAL) proteins
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This differential extraction protocol allows separation of FL from MAL proteins, which is crucial for understanding the protein's native state and associations. Verification of fraction purity should be performed using immunoblotting with markers for different chloroplast compartments (e.g., FKBP16-1 for soluble lumen, PsbO for membrane-associated lumen, D1 for thylakoid membrane, and ClpC for stroma) .
How can researchers effectively study protein-protein interactions involving the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein?
Several complementary approaches should be used to comprehensively characterize protein-protein interactions:
| Technique | Application | Advantages | Limitations |
|---|---|---|---|
| Yeast two-hybrid | Initial screening of interaction partners | High-throughput capability | May produce false positives, requires nuclear localization |
| Split-GFP or BIFC | In vivo verification of interactions | Visualizes interactions in plant cells | Requires protein tagging, may affect function |
| Co-immunoprecipitation | Isolation of protein complexes | Captures native complexes | Requires specific antibodies, may disrupt weak interactions |
| Blue native PAGE | Analysis of intact protein complexes | Preserves native structure | Limited resolution for large complexes |
| Crosslinking coupled with MS | Identification of transient interactions | Captures dynamic interactions | Complex data analysis, potential artifacts |
As demonstrated in research with TLP7.6 and CYP38, a combined approach starting with yeast two-hybrid screening followed by verification through multiple techniques provides the most reliable results. For thylakoid lumenal proteins, special consideration should be given to the unique environment of the lumen, including its pH fluctuations and restricted space, which may affect interaction properties .
What is the precise role of the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein in photosystem assembly or repair?
Based on studies of other thylakoid lumenal proteins, the 25.3 kDa protein may participate in photosystem assembly or repair processes. To determine its specific role, researchers should employ the following methodological approach:
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Generate knockout/knockdown lines in model systems (Arabidopsis if spinach is challenging)
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Characterize photosynthetic parameters under normal and stress conditions (e.g., high light, temperature stress)
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Assess photosystem assembly using blue native PAGE to visualize:
| Protein Complex | Expected MW | Function | Assessment Method |
|---|---|---|---|
| PSII Supercomplexes | 800-1,000 kDa | Efficient light harvesting and energy transfer | BN-PAGE, followed by 2D-PAGE |
| PSII Dimers | ~480 kDa | Core photosynthetic function | Immunoblotting for D1, D2 |
| PSII Monomers | ~280 kDa | Assembly intermediate | Pulse-chase labeling to track assembly |
| CP43-less PSII | ~220 kDa | Repair intermediate | Accumulation under high light |
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Perform time-resolved assembly studies using cycloheximide or chloramphenicol to block protein synthesis and monitor repair
This approach, similar to that used for TLP7.6, would reveal whether the protein functions in assembly, repair, or stabilization of photosystems, and under what conditions its function becomes critical .
How does the Spinacia oleracea Thylakoid lumenal 25.3 kDa protein respond to environmental stresses?
Environmental stresses significantly impact photosynthesis and may alter the expression, localization, or function of thylakoid lumenal proteins. A comprehensive analysis would include:
| Stress Condition | Experimental Approach | Expected Changes | Evaluation Methods |
|---|---|---|---|
| High light stress | Expose plants to 1000-1500 μmol m⁻² s⁻¹ | Altered expression, possible phosphorylation | qRT-PCR, phosphoproteomics |
| Temperature stress | Cold (4°C) or heat (40°C) treatment | Modified interaction network, structural changes | Co-IP under stress conditions, thermal shift assays |
| Drought | Controlled water limitation | Potential involvement in photoprotection | Chlorophyll fluorescence, NPQ measurements |
| Oxidative stress | H₂O₂ or methyl viologen treatment | Changes in redox state, possible thiol modifications | Redox proteomics, non-reducing gels |
For each condition, researchers should monitor both changes in the protein itself (abundance, modifications) and functional consequences (photosynthetic efficiency, energy dissipation, ROS production). Comparing responses in wild-type and knockout/knockdown lines would further elucidate the protein's role in stress adaptation .
How does the function of Spinacia oleracea Thylakoid lumenal 25.3 kDa protein compare with homologous proteins in model organisms?
A comprehensive comparative analysis requires assessment across multiple species:
| Organism | Homologous Proteins | Functional Conservation | Specialized Functions |
|---|---|---|---|
| Arabidopsis thaliana | TLP family proteins | Core functions in photosystem maintenance | Model for genetic studies |
| Chlamydomonas reinhardtii | Algal homologs | Evolutionary conserved domains | Unicellular photosynthesis model |
| Synechocystis sp. | Cyanobacterial ancestors | Primordial photosynthetic functions | Prokaryotic thylakoid system |
Methodologically, researchers should:
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Perform sequence alignment and phylogenetic analysis to identify conserved domains
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Conduct complementation studies by expressing the spinach protein in homolog-deficient mutants of model organisms
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Assess functional conservation using standardized photosynthetic measurements
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Identify species-specific adaptations through structural comparisons and domain analysis
Such comparative approaches provide evolutionary context and may reveal specialized adaptations in Spinacia oleracea related to its specific environmental niche .
What experimental approaches can resolve contradictory findings about thylakoid lumenal protein functions across different species?
When research findings about homologous proteins show discrepancies across species, several methodological approaches can help resolve contradictions:
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Standardized experimental conditions:
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Use defined growth parameters (light intensity, photoperiod, temperature)
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Consistent protein extraction and fractionation protocols
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Standardized activity assays with identical buffer compositions
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Heterologous expression studies:
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Express proteins from different species in a common background
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Assess functional complementation quantitatively
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Identify species-specific requirements for activity
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Domain swap experiments:
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Create chimeric proteins exchanging domains between species
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Map functional differences to specific protein regions
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Identify critical amino acid differences
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Systems biology approach:
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Compare interaction networks across species
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Identify compensatory mechanisms in different organisms
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Model the impact of differences in stoichiometry or regulation
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This systematic approach can distinguish between true functional differences and artifacts of experimental conditions or genetic backgrounds .
How can cryo-electron microscopy advance our understanding of Spinacia oleracea Thylakoid lumenal 25.3 kDa protein's structure and interactions?
Cryo-electron microscopy (cryo-EM) offers significant advantages for studying thylakoid lumenal proteins:
| Cryo-EM Application | Research Objective | Methodological Considerations | Expected Outcomes |
|---|---|---|---|
| Single particle analysis | High-resolution structure determination | Requires highly pure, homogeneous samples | 3D structure at near-atomic resolution |
| Subtomogram averaging | In situ structural analysis | Preserves native membrane context | Visualization of protein in thylakoid environment |
| In situ cryo-ET | Visualization of native protein distribution | Requires thin samples (< 500 nm) | Spatial organization within lumen |
| Time-resolved cryo-EM | Capture structural dynamics | Synchronized sample preparation | Conformational changes during activity |
For effective implementation:
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Optimize sample preparation with gentle fixation methods
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Use phase plates for improved contrast of small proteins
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Implement computational approaches to identify the protein within the complex thylakoid environment
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Combine with mass spectrometry to identify interaction partners in observed complexes
This approach would reveal not only the protein's structure but also its position relative to photosystems and other lumenal components, providing insight into its functional mechanism .
What are the cutting-edge genetic engineering approaches to study the function of Spinacia oleracea Thylakoid lumenal 25.3 kDa protein?
Advanced genetic approaches offer new possibilities for functional studies:
| Genetic Technique | Application | Advantages | Methodological Considerations |
|---|---|---|---|
| CRISPR/Cas9 editing | Precise gene knockout or modification | Targeted mutations, minimal off-targets | Design of efficient guide RNAs, delivery methods for spinach |
| Inducible expression systems | Temporal control of protein levels | Study acute effects of protein loss | Optimization of induction conditions in planta |
| Optogenetic control | Light-controlled protein activity | Spatial and temporal precision | Engineering light-responsive domains without affecting localization |
| Proximity labeling (BioID/TurboID) | In vivo identification of interacting proteins | Captures transient interactions | Ensuring enzymatic activity in lumen environment |
| Fluorescent protein tagging | Visualization of dynamics | Real-time observation in living cells | Proper folding of FPs in lumen environment |
As demonstrated in the recent TLP7.6 research, CRISPR/Cas9 technology can effectively generate knockout lines to study lumenal protein function. For spinach, which is less amenable to transformation than Arabidopsis, alternative approaches include:
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Virus-induced gene silencing (VIGS) for transient knockdown
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Heterologous expression in Arabidopsis followed by functional studies
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Complementation of Arabidopsis mutants with spinach genes
These advanced tools allow researchers to move beyond correlative observations to establish causative relationships and dissect complex regulatory networks involving thylakoid lumenal proteins .
