Recombinant Cicer arietinum ATP synthase subunit c, chloroplastic (atpH)

What is the function of ATP synthase subunit c in chloroplasts?

ATP synthase subunit c in chloroplasts forms part of the critical membrane-embedded F₀ motor that drives ATP synthesis. This subunit assembles into an oligomeric c-ring embedded in the thylakoid membrane. During photosynthesis, protons are translocated across this membrane along an electrochemical gradient, and the flow of these protons drives the rotation of the c-ring . This mechanical rotation is coupled to the F₁ head, where ATP synthesis occurs through rotary catalysis .

The c-subunit ring serves as the interface between the proton motive force and the ATP synthesizing machinery, with the number of c-subunits in the ring varying between different organisms. This variation affects the ratio of protons translocated to ATP molecules synthesized, which is inherently related to the organism's metabolism .

How is recombinant atpH typically expressed in laboratory settings?

Recombinant atpH is typically expressed in bacterial systems, with E. coli being the most common host organism. Due to the hydrophobic nature of this membrane protein, expression often employs fusion protein strategies to enhance solubility. A proven methodology involves expressing the hydrophobic atpH subunit as a soluble fusion protein, commonly with maltose binding protein (MBP) .

The process typically follows these steps:

• Gene insertion into a plasmid with codon optimization for the expression host

• Expression as a soluble MBP-atpH fusion protein

• Cleavage of the fusion protein to release the atpH subunit

• Purification via reversed-phase chromatography

This approach has been successfully demonstrated with spinach chloroplast ATP synthase c-subunit and can be adapted for Cicer arietinum, allowing the soluble expression of this eukaryotic membrane protein in BL21 derivative E. coli cells .

What structural characteristics define the ATP synthase subunit c in Cicer arietinum?

ATP synthase subunit c in Cicer arietinum, like other plant chloroplastic ATP synthase c-subunits, is characterized by an alpha-helical secondary structure . The protein typically contains two transmembrane alpha-helices connected by a hydrophilic loop region. The number of subunits in a functional c-ring varies among species, affecting the stoichiometry of proton translocation to ATP synthesis.

While specific structural data for Cicer arietinum atpH is limited, we can infer from related species that it likely features conserved amino acid residues essential for proton binding and translocation across the membrane. The protein would be expected to contain a crucial proton-binding site, typically involving a conserved carboxylate residue (often glutamate) in one of the transmembrane helices .

What expression systems are optimal for producing recombinant chloroplastic atpH?

For optimal expression of recombinant chloroplastic atpH, several expression systems can be considered, each with specific advantages:

• E. coli-based expression systems: The most widely used approach involves BL21 derivative E. coli cells with a plasmid containing a codon-optimized gene insert . This system offers high yield and relatively straightforward protocols. To overcome the hydrophobicity challenges, expressing atpH as a fusion protein with maltose binding protein (MBP) significantly enhances solubility .

• Yeast expression systems: For researchers studying post-translational modifications or requiring eukaryotic processing, yeast systems provide advantages while maintaining relatively high yields .

• Baculovirus expression systems: When higher eukaryotic protein folding is critical, baculovirus-infected insect cells can provide an appropriate environment, though with increased complexity and cost .

• Mammalian cell expression systems: For applications requiring mammalian-specific processing or interaction studies with mammalian proteins, these systems offer the most native-like production environment but with lower yields and higher costs .

The expression methodology should include optimization of induction temperature, inducer concentration, and expression duration to balance between protein yield and proper folding. For most research applications, the E. coli system with MBP fusion proves most efficient for chloroplastic atpH .

How can the purity of recombinant atpH be assessed?

Assessing the purity of recombinant atpH requires multiple complementary techniques due to its hydrophobic nature:

• SDS-PAGE analysis: The primary method for purity assessment, with professional standards requiring ≥85% purity as determined by densitometry of protein bands . For atpH, use specialized gel systems optimized for low molecular weight hydrophobic proteins.

• Western blotting: Use antibodies specific to atpH or epitope tags to verify the identity of the purified protein and detect potential degradation products.

• Mass spectrometry: LC-MS/MS analysis provides definitive identification and can detect post-translational modifications, contaminants, and verify the molecular mass of the purified protein.

• Circular dichroism (CD) spectroscopy: This technique verifies the correct alpha-helical secondary structure of the purified atpH, which is critical to confirm proper folding . CD spectra should show characteristic minima at 208 and 222 nm, typical of alpha-helical proteins.

• Size exclusion chromatography: Used to detect potential oligomerization or aggregation of the purified protein.

The combination of these techniques provides comprehensive assessment of both purity and structural integrity of the recombinant atpH protein .

What are the critical considerations for maintaining protein stability during atpH purification?

Maintaining stability of recombinant atpH during purification requires addressing several critical factors:

• Detergent selection: The hydrophobic nature of atpH necessitates careful detergent selection. Mild detergents like n-dodecyl-β-D-maltoside (DDM) or digitonin better preserve protein structure compared to more aggressive detergents like SDS.

• Temperature control: All purification steps should be performed at 4°C to minimize protein degradation and aggregation.

• Protease inhibition: Including a comprehensive protease inhibitor cocktail prevents degradation, especially after cleavage of fusion tags.

• pH optimization: Maintaining optimal pH (typically between 7.0-8.0) stabilizes the protein structure. pH extremes should be avoided except during specific purification steps like reversed-phase chromatography .

• Salt concentration: Including appropriate salt concentrations (typically 100-300 mM NaCl) shields electrostatic interactions and reduces aggregation.

• Reducing agents: Addition of reducing agents like DTT or β-mercaptoethanol (typically 1-5 mM) prevents oxidation of cysteine residues and potential aberrant disulfide bond formation.

• Glycerol addition: Including 5-10% glycerol in storage buffers enhances protein stability during long-term storage.

• Rapid processing: Minimizing the time between purification steps reduces opportunities for degradation or aggregation.

For chloroplastic atpH specifically, the purification methodology involving reversed-phase column chromatography after cleavage from the MBP fusion partner has proven effective in producing stable, properly folded protein .

How does the c-ring stoichiometry in Cicer arietinum compare to other plant species?

The c-ring stoichiometry (the number of c-subunits forming the oligomeric ring) is a critical parameter that determines the H⁺/ATP ratio during ATP synthesis. While specific data for Cicer arietinum is not yet comprehensively documented, we can draw insights from comparative analyses of ATP synthase across plant species.

The c-ring stoichiometry shows variability across organisms:

• Bacteria: 8-15 c-subunits

• Chloroplasts: typically 14 c-subunits (in spinach)

• Mitochondria: typically 8 c-subunits (in vertebrates)

This variability is believed to be a fundamental adaptation to different bioenergetic environments. For chloroplastic ATP synthase, the higher number of c-subunits correlates with the need to utilize the relatively small proton motive force generated during photosynthesis .

To determine the specific c-ring stoichiometry in Cicer arietinum, researchers should consider:

• Cryo-electron microscopy (cryo-EM): This has emerged as the definitive methodology for determining c-ring structure and stoichiometry, capable of resolving side chains of all protein subunits and the nucleotides in the F₁ head .

• Atomic force microscopy (AFM): Can provide complementary structural information on isolated c-rings.

• Mass determination through native mass spectrometry: Can determine the mass of the intact c-ring complex, allowing calculation of subunit number.

The stoichiometry directly influences the bioenergetic efficiency of the organism, with implications for understanding evolutionary adaptations in photosynthetic efficiency in legumes like Cicer arietinum .

What insights can recombinant atpH provide about proton translocation mechanisms?

Recombinant atpH serves as a powerful tool for investigating the molecular mechanisms of proton translocation across the thylakoid membrane. Several methodological approaches yield valuable insights:

• Site-directed mutagenesis studies: By creating specific mutations in the recombinant atpH and analyzing their effects on function, researchers can identify amino acid residues critical for proton binding and translocation. Key targets include the conserved carboxylate residue in the proton-binding site .

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique maps proton accessibility and exchange rates throughout the protein structure, revealing conformational dynamics associated with proton binding and release.

• Biophysical measurements: Techniques like solid-state NMR and infrared spectroscopy can probe the protonation states of key residues during the catalytic cycle.

• Reconstitution into liposomes: Purified recombinant atpH can be reconstituted into liposomes to measure proton translocation directly using pH-sensitive dyes or electrodes.

High-resolution structural studies using cryo-EM have successfully resolved the proton pathway to and from the rotor ring in chloroplast ATP synthase . These studies, combined with functional assays using recombinant atpH, demonstrate that proton translocation involves a series of conformational changes in the c-ring that drive rotation, coupling proton movement to ATP synthesis in the F₁ head .

How can site-directed mutagenesis of atpH reveal functional domains?

Site-directed mutagenesis of recombinant atpH provides a precise methodology for mapping functional domains and understanding structure-function relationships. The experimental approach should follow these steps:

• Target selection based on sequence conservation: Analyze sequence alignments across species to identify highly conserved residues that likely play crucial functional roles .

• Structural context analysis: Use available structural data from related species to identify residues in key positions, such as:

  • Proton-binding site residues

  • Residues at the interface with other subunits

  • Residues involved in the rotation mechanism

  • Transmembrane helix packing residues

• Systematic mutagenesis strategy:

  • Conservative mutations (e.g., D→E) to test functional importance

  • Non-conservative mutations to disrupt function (e.g., D→A)

  • Cysteine scanning mutagenesis to map accessibility

  • Introduction of reporter groups for spectroscopic studies

• Functional assays:

  • ATP synthesis/hydrolysis activity measurements

  • Proton translocation assays

  • Assembly analysis using native gel electrophoresis

  • Rotational measurements using single-molecule techniques

• Structural confirmation:

  • Circular dichroism to verify proper folding

  • Limited proteolysis to assess structural integrity

  • Thermal stability assays to measure effects on protein stability

Key insights from mutagenesis studies of ATP synthase subunit c have revealed critical functional domains, including the proton-binding site, the interface with the peripheral stalk, and regions involved in c-ring assembly. These studies also help understand how the c-ring stoichiometry is determined at the molecular level, which remains one of the major unresolved questions in ATP synthase research .

What are common challenges in expressing functional recombinant atpH?

Expressing functional recombinant atpH presents several challenges that researchers commonly encounter. Here are the major issues and methodological solutions:

• Low expression yields:

  • Cause: Hydrophobicity and potential toxicity to host cells

  • Solution: Use fusion proteins like MBP to enhance solubility , codon optimization for expression host, lower induction temperature (16-18°C), and reduced inducer concentration

• Inclusion body formation:

  • Cause: Improper folding of hydrophobic membrane protein

  • Solution: Express as fusion protein, optimize expression conditions, or develop refolding protocols from inclusion bodies using mild detergents

• Proteolytic degradation:

  • Cause: Recognition by host proteases

  • Solution: Use protease-deficient host strains, include protease inhibitors, optimize purification speed

• Improper folding:

  • Cause: Membrane proteins require specific lipid environments

  • Solution: Verify alpha-helical structure using CD spectroscopy , screen different detergents for extraction and purification

• Loss of function after purification:

  • Cause: Disruption of native structure during detergent solubilization

  • Solution: Reconstitute purified protein into liposomes or nanodiscs to restore a membrane-like environment

• Oligomerization issues:

  • Cause: Hydrophobic surfaces promote non-specific aggregation

  • Solution: Use size exclusion chromatography to isolate properly assembled complexes, optimize detergent:protein ratios

For chloroplastic atpH specifically, the development of a recombinant bacterial expression and column purification system has successfully addressed many of these challenges, allowing the production of significant quantities of highly purified c1 subunit with the correct alpha-helical secondary structure .

How can contradictory results in atpH activity assays be reconciled?

Contradictory results in atpH activity assays are commonly encountered in research and require systematic investigation to reconcile. Here's a methodological approach to address discrepancies:

• Standardize protein preparation:

  • Ensure consistent purification protocols

  • Verify protein integrity through SDS-PAGE, western blotting, and mass spectrometry

  • Confirm proper folding using CD spectroscopy to detect alpha-helical structure

  • Quantify protein concentration using multiple methods (Bradford, BCA, amino acid analysis)

• Control assay conditions rigorously:

  • Document and standardize buffer composition, pH, salt concentration

  • Control temperature precisely during measurements

  • Use internal standards to normalize between experimental runs

  • Consider the effects of different detergents on activity

• Address methodological differences:

  • Compare different activity assay methods (ATPase activity, proton translocation, rotational assays)

  • Calibrate instruments regularly

  • Use multiple technical and biological replicates

  • Blind sample testing to eliminate unconscious bias

• Investigate biological variables:

  • The activity of ATP synthase shows pH dependence

  • Redox regulation affects ATP synthase activity in plants (autoinhibition in the dark via a β-hairpin redox switch in subunit γ)

  • ATP synthase can reverse its function depending on conditions, acting as ATPase instead of ATP synthase

• Data analysis approaches:

  • Use statistical methods appropriate for the data distribution

  • Consider Bayesian analysis for integrating prior knowledge with new data

  • Meta-analysis techniques to combine results from multiple experiments

  • Develop mathematical models to explain apparent contradictions

Remember that native ATP synthase contains regulatory mechanisms, such as the inhibitor protein IF₁, which inhibits ATP hydrolysis in a pH-dependent manner when mitochondrial membrane potential drops . Similar regulatory mechanisms might affect chloroplastic ATP synthase activity assays.

What statistical approaches are recommended for analyzing ATP synthase kinetics?

Analyzing ATP synthase kinetics requires robust statistical approaches to accurately interpret experimental data. Here are recommended statistical methodologies specifically for atpH research:

• Enzyme kinetics modeling:

  • Apply Michaelis-Menten kinetics to determine Km and Vmax parameters

  • Use non-linear regression rather than linear transformations (e.g., Lineweaver-Burk)

  • Consider more complex models for regulatory effects or inhibition patterns

  • Analyze cooperativity effects using Hill equation

• Time series analysis:

  • Implement repeated measures ANOVA for time-dependent assays

  • Use regression models with time as a continuous variable

  • Consider non-linear models for complex time-dependent behavior

• Comparative analysis across conditions:

  • Two-way ANOVA to assess interactions between factors (e.g., pH and temperature)

  • Post-hoc tests with appropriate correction for multiple comparisons (e.g., Tukey HSD)

  • Effect size calculation (Cohen's d or η²) to quantify magnitude of differences

• Quality control and outlier detection:

  • Grubbs' test or Dixon's Q-test for identifying outliers

  • Shapiro-Wilk test to verify normality assumptions

  • Levene's test to check homogeneity of variance

• Advanced statistical approaches:

  • Mixed-effects models to account for random and fixed effects

  • Bootstrap resampling for robust parameter estimation

  • Bayesian inference for incorporating prior knowledge

  • Principal component analysis for exploring multivariate data

• Reporting standards:

  • Report mean ± standard deviation or standard error

  • Include 95% confidence intervals for key parameters

  • Report exact p-values rather than thresholds

  • Include power analysis to justify sample sizes

For evolutionary rate analysis of ATP synthase genes, the Ka/Ks ratio (nonsynonymous to synonymous substitution rates) can be calculated to identify selection pressures. Data shows that ATP synthase genes are under purifying selection (Ka/Ks = 0.1337), indicating evolutionary conservation of function .

How is atpH being used to study evolutionary relationships among legumes?

ATP synthase subunit c (atpH) serves as a valuable molecular marker for evolutionary studies among legumes due to its essential function and evolutionary conservation. Current methodological approaches in this research frontier include:

• Chloroplast genome sequencing and comparative genomics:

  • Complete chloroplast genome sequencing of Cicer arietinum and related legumes

  • Annotation and identification of atpH and other ATP synthase genes

  • Comparative analysis of gene order and structure across species

• Evolutionary rate analysis:

  • Calculation of synonymous (Ks) and nonsynonymous (Ka) substitution rates

  • Determination of Ka/Ks ratios to assess selection pressure

  • ATP synthase genes typically show low Ka/Ks ratios (average 0.1337), indicating strong purifying selection and functional conservation

• Phylogenetic reconstruction:

  • Multiple sequence alignment of atpH sequences across legume species

  • Construction of phylogenetic trees using maximum likelihood, Bayesian inference, or neighbor-joining methods

  • Estimation of divergence times using molecular clock approaches

• Codon usage analysis:

  • Assessment of codon bias in atpH genes across species

  • Correlation of codon usage with gene expression levels

  • Most chloroplast genes show preference for codons ending with A/T rather than C/G (RSCU ≥ 1 for A/T-ending codons)

• RNA editing site comparison:

  • Identification and comparison of RNA editing sites in atpH transcripts

  • Analysis of conservation of editing sites across legume species

  • Most RNA editing in chloroplast genes converts cytidine to uridine, often resulting in serine to leucine amino acid changes

This research contributes to understanding the evolutionary history of legumes, including Cicer arietinum, and provides insights into the adaptation of photosynthetic machinery across different ecological niches.

What role might atpH play in engineering plants with enhanced photosynthetic efficiency?

ATP synthase subunit c (atpH) represents a key target for engineering enhanced photosynthetic efficiency in plants through several methodological approaches:

• Optimization of c-ring stoichiometry:

  • Engineering the number of c-subunits in the ring alters the H⁺/ATP ratio

  • Decreasing the number of c-subunits could theoretically improve ATP synthesis efficiency by requiring fewer protons per ATP

  • This requires precise genetic engineering of the atpH gene and assembly factors

• Modification of proton-binding residues:

  • Engineering the proton-binding site to optimize proton affinity and release kinetics

  • Site-directed mutagenesis of key residues to adjust pKa values

  • Potential to fine-tune the pH dependency of ATP synthase activity

• Redox regulation optimization:

  • Modification of the redox regulatory mechanism (β-hairpin redox switch in subunit γ)

  • Engineering faster activation upon illumination or reduced inhibition in low light

  • This could improve ATP production during fluctuating light conditions

• Enhancement of ATP synthase stability:

  • Engineering increased thermal stability for better performance under heat stress

  • Improving assembly efficiency through optimization of atpH interaction interfaces

  • Reducing photoinhibition-related damage to the ATP synthase complex

• Metabolic coupling improvement:

  • Engineering the NADH-dependent regulation mechanism that controls ATP synthase

  • Optimization of the interaction between apoptosis-inducing factor 1 (AIFM1) and adenylate kinase 2 (AK2) as gatekeepers of ATP synthase

  • This could improve metabolic adaptation to fluctuating energy availability

Current methodological challenges include ensuring proper assembly of the modified ATP synthase complex, avoiding unintended consequences for photosynthetic electron transport, and maintaining appropriate regulation of the ATP/ADP ratio. Engineering approaches must consider the whole-plant context and avoid disrupting the delicate balance between different photosynthetic processes .

How do post-translational modifications affect atpH function?

Post-translational modifications (PTMs) of ATP synthase subunit c (atpH) represent an emerging area of research with significant implications for understanding regulation and function. Current methodological approaches in this field include:

• Identification of PTMs:

  • Mass spectrometry-based proteomics (MS/MS) to identify modifications

  • Enrichment strategies for specific modifications (e.g., phosphopeptide enrichment)

  • Site-specific antibodies for detecting common modifications

  • Top-down proteomics to analyze intact proteins with modifications

• Functional characterization of PTMs:

  • Site-directed mutagenesis to create non-modifiable variants (e.g., S→A for phosphorylation sites)

  • Creation of phosphomimetic mutations (e.g., S→D or S→E)

  • In vitro modification using purified enzymes followed by activity assays

  • Development of assays to measure effects on c-ring assembly, stability, and rotation

• Regulation of PTMs:

  • Identification of enzymes responsible for adding or removing modifications

  • Analysis of modification dynamics under different physiological conditions

  • Investigation of light/dark, stress, or developmental regulation of modifications

  • Study of redox-dependent modifications in response to changing chloroplast redox state

• PTM interplay with protein-protein interactions:

  • Analysis of how modifications affect interactions with other ATP synthase subunits

  • Investigation of potential interactions with regulatory proteins

  • Study of the NADH-dependent interaction between AIFM1 and AK2 as regulators of ATP synthase activity

• Conservation of modification sites:

  • Comparative analysis of potential modification sites across species

  • Correlation of site conservation with functional importance

  • Evolutionary analysis of modification enzymes