Recombinant Daucus carota ATP synthase subunit c, chloroplastic (atpH)

What is Recombinant Daucus carota ATP synthase subunit c, chloroplastic (atpH)?

Recombinant Daucus carota ATP synthase subunit c is a chloroplastic protein component of the ATP synthase complex derived from carrots. This protein is also referred to as ATP synthase F(0) sector subunit c, ATPase subunit III, F-type ATPase subunit c, or Lipid-binding protein. The gene encoding this protein is designated as atpH, and its Uniprot accession number is Q0G9X5 . This protein plays a crucial role in energy transduction within chloroplasts, facilitating ATP synthesis through proton transport across membranes during photosynthesis. As a recombinant protein, it is produced using expression systems that allow for the study of its structure, function, and interactions outside of its native cellular environment.

What is the complete amino acid sequence of Daucus carota ATP synthase subunit c?

The complete amino acid sequence of Daucus carota ATP synthase subunit c, chloroplastic (atpH) consists of 81 amino acids as follows:

MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV

This sequence contains a high proportion of hydrophobic amino acids, particularly in membrane-spanning regions, which is characteristic of proteins embedded in lipid bilayers. The protein's expression region spans residues 1-81, representing the full-length protein . Understanding this sequence is fundamental for researchers conducting structural analyses, designing mutagenesis experiments, or developing antibodies against specific epitopes of the protein.

How does the structure of ATP synthase subunit c relate to its function in chloroplasts?

ATP synthase subunit c forms a crucial component of the F0 portion of the ATP synthase complex, which is embedded in the thylakoid membrane of chloroplasts. The protein adopts a hairpin-like structure with two membrane-spanning α-helices connected by a short loop. Multiple c-subunits assemble into a ring structure within the membrane, forming a proton channel. The amino acid sequence reveals hydrophobic residues (like alanine, leucine, and valine) that facilitate membrane insertion and stability, while strategically positioned polar residues enable proton translocation .

The functional mechanism involves proton movement through the c-ring, driven by the electrochemical gradient established during photosynthesis. This rotation is mechanically coupled to conformational changes in the F1 portion of ATP synthase, driving ATP synthesis. The specific composition of the carrot ATP synthase c-subunit likely reflects evolutionary adaptations to optimize photosynthetic efficiency under the plant's native growing conditions.

What expression systems are most effective for producing recombinant Daucus carota atpH protein?

Based on research protocols with similar chloroplastic proteins, the most effective expression systems for producing functional recombinant Daucus carota atpH include:

• E. coli-based systems: Studies demonstrate that E. coli strains modified to express small heat shock proteins from carrot (like DcHsp17.7) show enhanced protein accumulation and stability . When expressing membrane proteins like ATP synthase subunit c, E. coli strains with reduced protease activity (like BL21(DE3)) coupled with tightly regulated promoters (T7 or tac) typically yield better results.

• Transgenic cell line approach: Creating stable transgenic E. coli lines expressing carrot chaperone proteins significantly improves recombinant protein yields. Research shows that such lines accumulated approximately 130% more cellular proteins under stress conditions compared to control lines .

• Cold-induction protocols: Expression at reduced temperatures (16°C) has been shown to increase protein folding efficiency and reduce inclusion body formation for membrane proteins like ATP synthase components .

When optimizing expression, key factors include codon optimization for the host organism, use of fusion tags to enhance solubility (while considering their potential impact on structure and function), and careful selection of induction conditions to balance protein yield with proper folding.

What are the recommended storage conditions for maintaining stability of recombinant atpH protein?

For optimal stability of recombinant Daucus carota ATP synthase subunit c, the following storage conditions are recommended:

• Storage buffer composition: The protein should be stored in a Tris-based buffer with 50% glycerol, specifically optimized for this protein's stability .

• Temperature conditions: For short-term storage (up to one week), aliquots can be maintained at 4°C. For extended storage, -20°C is suitable, while critical samples should be stored at -80°C .

• Freeze-thaw considerations: Repeated freezing and thawing should be strictly avoided as it leads to protein denaturation and activity loss. Working aliquots should be prepared during initial storage to minimize freeze-thaw cycles .

• Additives for enhanced stability: Depending on experimental requirements, stabilizing agents such as reducing agents (DTT or β-mercaptoethanol at 1-5 mM) may be added to prevent oxidation of cysteine residues, though their necessity should be determined experimentally for this specific protein.

When handling the protein, researchers should maintain sterile conditions to prevent microbial contamination and process samples on ice whenever possible to minimize degradation.

How does ATP synthase function relate to carotenoid metabolism in carrots?

Research reveals a complex relationship between ATP synthase function and carotenoid metabolism in carrots, particularly under varying environmental conditions:

• Energy coupling: ATP synthase provides the essential energy currency (ATP) required for numerous enzymatic reactions in carotenoid biosynthesis pathways. RNA sequencing studies identified 20 genes related to carotenoids among 482 differentially expressed genes in carrots under CO2 enrichment, many involved in either carotenoid biosynthesis or photosystem membrane proteins .

• Coordinated regulation: Gene expression analysis reveals that ATP production capacity and carotenoid biosynthesis are co-regulated in response to environmental stimuli. Under elevated CO2 conditions, carrot plants showed increased expression of genes involved in both ATP synthesis and carotenoid metabolism .

• Metabolic impacts: Enhanced ATP synthase activity correlates with increased carotenoid production. Experimental evidence demonstrates that CO2 enrichment significantly increased both α-carotene and β-carotene contents in carrot taproots, with β-carotene contents rising from 232.89 ± 1.88 μg·g-1 FW under ambient CO2 to 792.76 ± 6.92 μg·g-1 FW under elevated CO2 .

This relationship suggests that optimization of ATP synthase function could be a target for enhancing nutritional quality in carrots through increased carotenoid production, particularly in controlled growth environments where CO2 levels can be manipulated.

What impacts do environmental stressors have on atpH expression and function?

Environmental stressors significantly modulate atpH expression and function in Daucus carota, with important implications for photosynthetic efficiency and plant adaptation:

• CO2 concentration effects: Elevated CO2 levels significantly alter the expression of chloroplastic genes, including those encoding ATP synthase components. Studies demonstrate that under CO2 enrichment, carrot plants show upregulation of photosystem membrane proteins, leading to increased biomass and carotenoid content . This suggests that atpH expression may be enhanced under elevated CO2 conditions as part of the plant's adaptation to optimize energy capture.

• Heat stress response: Heat stress triggers protective mechanisms that indirectly affect ATP synthase function. Research with carrot heat shock proteins shows that under stress conditions, expression of protective proteins increases to maintain cellular function. Transgenic expression of carrot DcHsp17.7 in E. coli demonstrated improved growth and cell viability under heat stress conditions , suggesting similar protective mechanisms may operate in carrot chloroplasts to maintain ATP synthase function during thermal stress.

• Acetate and pH stress resilience: Studies indicate that stress-protective proteins from carrot (like DcHsp17.7) can significantly improve cellular tolerance to acetate and alkaline conditions . This suggests that carrot chloroplasts have evolved robust adaptive mechanisms to maintain ATP synthase function across varying environmental conditions.

Understanding these environmental response mechanisms provides valuable insights for optimizing growth conditions in both research and agricultural settings, particularly as climate change introduces more variable growing conditions.

How can researchers effectively measure the enzymatic activity of carrot ATP synthase?

Measuring the enzymatic activity of carrot ATP synthase requires specialized techniques addressing both ATP synthesis and hydrolysis directions:

• ATP synthesis assay:

  • Isolated chloroplast method: Isolate intact chloroplasts from carrot leaves using differential centrifugation in sorbitol-based isolation buffer.

  • Light-dependent ATP synthesis: Measure ATP production by monitoring luciferin-luciferase luminescence after illuminating chloroplasts in the presence of ADP and inorganic phosphate.

  • Inhibitor controls: Use specific inhibitors like oligomycin or DCCD (N,N'-dicyclohexylcarbodiimide) as negative controls to confirm ATP synthase-specific activity.

• ATP hydrolysis assay:

  • Phosphate release measurement: Quantify inorganic phosphate released during ATP hydrolysis using malachite green or molybdate-based colorimetric assays.

  • Coupled enzyme assay: Monitor ATP hydrolysis by coupling ADP production to NADH oxidation via pyruvate kinase and lactate dehydrogenase, measuring absorbance decrease at 340 nm.

• Proton pumping assay:

  • pH indicator method: Track proton movement using pH-sensitive fluorescent dyes like ACMA (9-amino-6-chloro-2-methoxyacridine) when the enzyme runs in reverse.

  • Membrane potential measurement: Use voltage-sensitive dyes to monitor membrane potential changes associated with proton translocation.

When working with recombinant ATP synthase subunit c specifically, researchers should consider reconstitution approaches where the purified protein is incorporated into liposomes to recreate a functional membrane environment, enabling more accurate activity measurements.

What approaches can resolve low expression yields of recombinant atpH protein?

Researchers facing low expression yields of recombinant Daucus carota ATP synthase subunit c can implement several strategic approaches:

• Co-expression with chaperone proteins: Evidence from studies with carrot heat shock proteins suggests significant benefits from co-expression strategies. Transgenic cell lines expressing DcHsp17.7 accumulated approximately 130% more cellular proteins than control lines under stress conditions . For membrane proteins like ATP synthase subunit c, co-expression with appropriate chaperones can dramatically improve proper folding and stability.

• Optimization of expression conditions:

  • Temperature modification: Reducing expression temperature to 16°C has demonstrated 2.2-fold higher recombinant protein accumulation in cells expressing carrot chaperones compared to control conditions .

  • Induction protocol adjustments: Gradually increasing inducer concentration rather than single-dose addition can improve membrane protein expression.

  • Media composition: Supplementing with specific additives like glycerol (0.5-2%) or specific amino acids can enhance membrane protein yields.

• Expression vector and fusion partner selection:

  • Fusion tags: Strategic use of solubility-enhancing tags (like SUMO, MBP, or TrxA) can improve initial expression.

  • Signal sequence optimization: For membrane proteins, careful selection of signal sequences appropriate for the expression system is critical.

• Stress response mitigation: Research demonstrates that in the presence of stressors like acetate, cells expressing carrot heat shock proteins maintained high recombinant protein levels while control cells showed 70% reduction . Implementing strategies to mitigate cellular stress during expression can therefore significantly improve yields.

Systematic optimization of these parameters, preferably using a design of experiments (DOE) approach, typically yields the best results for challenging membrane proteins like ATP synthase components.

What analytical techniques are most informative for studying structure-function relationships of recombinant atpH?

Multiple analytical techniques provide complementary insights into the structure-function relationships of recombinant Daucus carota ATP synthase subunit c:

• Spectroscopic methods:

  • Circular dichroism (CD) spectroscopy: Essential for analyzing secondary structure content and stability of the protein under varying conditions.

  • Fluorescence spectroscopy: Valuable for monitoring conformational changes when intrinsic fluorophores (tryptophan, tyrosine) are present or when using environmentally sensitive fluorescent probes.

• Structural biology approaches:

  • X-ray crystallography: Though challenging for membrane proteins, provides atomic-level structural details when successful.

  • Cryo-electron microscopy: Increasingly powerful for membrane protein complexes, potentially revealing the arrangement of c-subunits in the complete ATP synthase complex.

  • NMR spectroscopy: Suitable for analyzing dynamics and localized structural features, particularly with selective isotopic labeling.

• Functional analyses:

  • Site-directed mutagenesis: Systematic mutation of key residues followed by activity assays can reveal functional roles of specific amino acids. The provided sequence information serves as the foundation for designing these mutations.

  • Reconstitution studies: Incorporating the protein into liposomes of defined composition allows controlled investigation of proton transport and ATP synthesis activities.

• Biophysical characterization:

  • Thermal shift assays: Assess protein stability under varying conditions.

  • Surface plasmon resonance: Measure interaction with other ATP synthase components or regulatory molecules.

Integration of multiple techniques provides the most comprehensive understanding, as each method offers unique insights into different aspects of structure-function relationships.

How does carrot ATP synthase compare with other plant species in structure and function?

Comparative analysis of ATP synthase subunit c across plant species reveals both evolutionary conservation and species-specific adaptations:

• Sequence conservation patterns: The carrot ATP synthase subunit c has the characteristic hydrophobic profile typical of membrane-embedded c-subunits across species. While the central ion-binding site residues show high conservation, variations occur particularly in the N-terminal region and connecting loop, potentially reflecting adaptations to specific environmental niches .

• Functional variations: Comparative studies suggest that while the fundamental mechanism of proton translocation coupled to ATP synthesis is conserved, kinetic properties vary between species. These differences likely reflect adaptations to different photosynthetic demands, with carrot showing adaptations related to its root storage function and relatively high carotenoid production capacity .

• Regulatory distinctions: Expression patterns of ATP synthase components show species-specific regulation. Unlike some other plants, carrot shows particularly strong responses to CO2 enrichment, with coordinated upregulation of photosynthetic machinery including ATP synthase components, which correlates with increased carotenoid production .

• Structural adaptations: While the basic c-ring architecture is conserved, subtle variations in c-subunit structure between species affect proton-to-ATP ratios and energy conversion efficiency. These adaptations likely reflect evolutionary optimization for specific environmental conditions.

This comparative understanding has implications for both basic research and applied agricultural science, potentially informing strategies to enhance photosynthetic efficiency across diverse crop species.

What are the implications of atpH research for improving stress tolerance in crops?

Research on ATP synthase subunit c and related proteins in Daucus carota provides valuable insights for crop improvement strategies:

The translational potential of this research is particularly relevant in the context of climate change, where crops face increasingly variable growing conditions and multiple simultaneous stressors.

What are the best practices for purifying recombinant Daucus carota ATP synthase subunit c?

Purification of recombinant Daucus carota ATP synthase subunit c requires specialized approaches for membrane proteins:

• Affinity chromatography optimization:

  • Tag selection: While various tags are possible, recombinant expressions commonly utilize a His-tag approach that enables purification via Ni-NTA affinity chromatography .

  • Buffer composition: Purification buffers should contain mild detergents (e.g., n-dodecyl-β-D-maltoside or CHAPS) at concentrations above their critical micelle concentration to maintain protein solubility while avoiding denaturation.

  • Imidazole gradient: For His-tagged proteins, a shallow imidazole gradient often yields better separation than step elution.

• Membrane protein-specific considerations:

  • Initial extraction: Effective solubilization from membranes requires optimization of detergent type and concentration, with careful consideration of the impact on protein structure and function.

  • Detergent exchange: During purification, it may be beneficial to transition from stronger solubilizing detergents to milder ones that better preserve native structure.

• Quality control measures:

  • Homogeneity assessment: Size exclusion chromatography provides critical information about protein aggregation state and homogeneity.

  • Functional verification: Activity assays should be performed at multiple purification stages to monitor retention of functional properties.

  • Storage optimization: Once purified, the protein should be stored in Tris-based buffer with 50% glycerol at -20°C for extended storage, with aliquots at 4°C for short-term use .

Successful purification requires balancing extraction efficiency with preservation of native structure and function, often necessitating empirical optimization of multiple parameters.

How can researchers effectively study the role of ATP synthase in carotenoid metabolism in carrots?

Investigating the relationship between ATP synthase and carotenoid metabolism requires integrated experimental approaches:

• Gene expression correlation analysis:

  • RNA-Seq methodology: Transcriptome sequencing has successfully identified differentially expressed genes related to both ATP synthase components and carotenoid metabolism under varying conditions .

  • qRT-PCR validation: Key findings from broader transcriptomic studies should be validated using targeted qRT-PCR of specific genes, including atpH and carotenoid biosynthesis enzymes like β-carotene hydroxylase.

• Metabolic flux analysis:

  • Isotope labeling: Using 13C-labeled precursors can track carbon flow through carotenoid biosynthetic pathways under conditions of altered ATP synthase activity.

  • ATP/ADP ratio measurement: Quantifying energy charge in different cellular compartments provides crucial information on energy availability for carotenoid synthesis.

• Experimental manipulation approaches:

  • Inhibitor studies: Specific inhibitors of ATP synthase (like oligomycin) can help establish causal relationships between ATP synthesis and carotenoid accumulation.

  • Genetic modification: Creating plants with altered expression of ATP synthase components can directly test their impact on carotenoid metabolism.

• Analytical methods for carotenoid quantification:

  • HPLC analysis: High-performance liquid chromatography enables precise quantification of different carotenoids, as demonstrated in studies measuring lutein, zeaxanthin, α-carotene, and β-carotene contents in carrot tissues .

  • Spectrophotometric approaches: For total carotenoid content, extraction with 80% acetone followed by absorbance measurement at 450 nm provides a reliable quantification method .

Integration of these approaches provides the most comprehensive understanding of how ATP synthase function influences carotenoid metabolism in carrots.

What are the most promising future research areas involving Daucus carota ATP synthase?

Several high-potential research directions emerge from current understanding of Daucus carota ATP synthase:

These research directions hold promise not only for advancing fundamental understanding of bioenergetics but also for developing practical applications in crop improvement and biotechnology as global challenges like climate change and food security become increasingly urgent.