Recombinant Phaseolus vulgaris ATP synthase subunit c, chloroplastic (atpH)

What is the basic structure and function of ATP synthase subunit c in Phaseolus vulgaris?

ATP synthase subunit c in Phaseolus vulgaris is a small, hydrophobic protein component of the FoF1-ATP synthase complex located in the chloroplast membrane. It forms part of the c-ring structure within the Fo domain that rotates during ATP synthesis. Each c-subunit contains a critical glutamic acid residue that participates in proton translocation across the membrane. This proton movement drives the rotation of the c-ring, which couples to the F1 domain to catalyze ATP synthesis. In chloroplasts, the ATP synthase harnesses the proton gradient established during photosynthesis to generate ATP, making it essential for plant energy metabolism .

How does the Phaseolus vulgaris atpH gene compare to other plant species?

The atpH gene in Phaseolus vulgaris encodes the chloroplastic ATP synthase subunit c. Chloroplast genes like atpH show relatively low nucleotide diversity within species, typically around 0.002 as observed in similar systems . The gene sequence contains AT-rich regions, with AT content ranging from 68.8% to 70.7% in analogous systems . Comparative genomic studies indicate that the atpF-atpH spacer region can be used for genetic identification purposes, though it demonstrates high conservation across varieties within a species. This conservation reflects the essential nature of ATP synthase function and the selective pressure to maintain its structure across evolutionary time .

What expression systems are most suitable for producing recombinant Phaseolus vulgaris ATP synthase subunit c?

For recombinant expression of Phaseolus vulgaris ATP synthase subunit c, Escherichia coli expression systems under the control of inducible promoters (such as lac) have proven effective for similar proteins . When expressing membrane proteins like ATP synthase subunit c, consideration must be given to the hydrophobic nature of the protein. Expression protocols typically involve optimization of growth temperature (often lowered to 18-25°C after induction), induction conditions, and addition of specific detergents for extraction. Purification generally requires acid extraction followed by multiple high-performance liquid chromatography (HPLC) steps to isolate the protein to homogeneity . It's crucial to verify both the immunological identity and bioactivity of the recombinant protein compared to native standards .

What are the most effective methods for isolating and purifying recombinant ATP synthase subunit c from expression systems?

The isolation and purification of recombinant ATP synthase subunit c requires specialized protocols due to its hydrophobic nature. The most effective methodology involves:

• Cell lysis under acidic conditions (pH 2-3) to extract the protein from membranes

• Initial purification through size exclusion chromatography

• Further purification using reverse-phase HPLC with a gradient of acetonitrile in trifluoroacetic acid

• Final polishing step with ion-exchange chromatography

This multi-step process typically yields three major forms of the recombinant protein: the correctly processed form, a formyl-methionyl form (with the initiating formyl-methionine still attached), and potentially truncated versions . It's critical to separate these forms for functional studies, as the formyl-methionyl version may have significantly reduced bioactivity (approximately 10% of the correctly processed form) while truncated versions are typically inactive .

How can researchers verify the structural integrity of recombinant Phaseolus vulgaris ATP synthase subunit c?

Verification of structural integrity requires a multi-faceted approach:

• Amino acid analysis to confirm the expected composition

• N-terminal sequencing to identify the presence or absence of formyl-methionine and correct processing

• Mass spectrometry to confirm the molecular weight and identify any post-translational modifications

• Circular dichroism spectroscopy to verify secondary structure elements

• Immunological assays using antibodies against the native protein to confirm structural epitopes

These methods collectively provide confidence in the structural fidelity of the recombinant protein before proceeding to functional studies. Additionally, NMR spectroscopy can provide atomic-level structural information for smaller membrane proteins like subunit c, though this requires isotopic labeling of the recombinant protein .

What functional assays can be used to assess the activity of recombinant ATP synthase subunit c?

Functional assessment of recombinant ATP synthase subunit c requires its integration into appropriate systems for measuring ATP synthase activity. Key assays include:

• Reconstitution assays: The purified subunit c can be reconstituted with other purified subunits to form a functional FoF1 complex in proteoliposomes.

• ATP synthesis activity: Measuring ATP synthesis driven by an artificially imposed proton gradient in the reconstituted system.

• ATP hydrolysis activity: Measuring the reverse reaction (ATP hydrolysis coupled to proton pumping).

• Proton translocation assays: Using pH-sensitive fluorescent dyes to directly measure proton movement across the membrane.

• Adenylate cyclase assays: For indirect measurement of functional activity in cellular systems .

It's essential to compare the activity of the recombinant protein with native standards or well-characterized control samples. Additionally, mutation studies targeting key residues (such as the conserved glutamic acid) can provide valuable insights into structure-function relationships .

How can researchers use the atpF-atpH spacer region for genetic identification of Phaseolus vulgaris varieties?

The atpF-atpH spacer region can serve as an effective DNA barcode for genetic identification of Phaseolus vulgaris varieties, similar to its application in other plant species. Researchers should:

• Extract total DNA from leaf tissue using standard protocols

• Amplify the atpF-atpH region using universal primers (e.g., atpF 5'-ACTCGCACACACTCCCTTTCC-3' and atpH 5'-GCTTTTATGGAAGCTTTAACAAT-3')

• Sequence the amplified products using Sanger sequencing

• Analyze sequence variation through:

  • Calculation of nucleotide diversity (π)

  • Haplotype analysis

  • Genetic distance estimation using appropriate models (e.g., Tamura 3-parameter)

While this region typically shows low nucleotide diversity within species (around 0.0019), it can still provide sufficient resolution for variety identification when combined with other chloroplast markers . The analysis should include estimation of GC/AT content ratios, which typically show AT-richness (68.8-70.7%) in this region .

What evolutionary insights can be gained from studying ATP synthase subunit c across different plant species?

ATP synthase subunit c is highly conserved across plant species due to its critical role in energy metabolism. Evolutionary studies can reveal:

• Conservation patterns: Identification of invariant residues essential for function versus variable regions that may relate to species-specific adaptations.

• Selective pressure: Analysis of synonymous versus non-synonymous substitutions can reveal the strength and direction of selection.

• Co-evolution: Examination of how mutations in subunit c correlate with changes in other ATP synthase subunits can reveal functional coupling between components.

• Adaptation mechanisms: Comparison of sequences from plants adapted to different environmental conditions may reveal how ATP synthase has evolved to function optimally under various stresses.

The low nucleotide diversity observed in chloroplast genes like atpH (π=0.0019) indicates strong purifying selection . Comprehensive phylogenetic analysis typically shows clustering by taxonomic groups, reflecting the early divergence of this essential machinery during plant evolution.

How do mutations in key residues of ATP synthase subunit c affect its function?

Mutations in key residues of ATP synthase subunit c can dramatically influence its function. Research on similar systems reveals that:

• Mutations in the conserved glutamic acid residue (analogous to E56 in some bacterial systems) significantly impact proton translocation. For example, substitution with aspartic acid (E→D) retains partial function but reduces ATP synthesis capacity by affecting proton uptake kinetics .

• The impact of mutations depends on their position within the c-ring. The efficiency of proton translocation decreases as mutated subunits are positioned farther apart around the c-ring, indicating cooperative interactions between adjacent subunits .

• Multiple mutations have cumulative effects. Systems with two E→D mutations show greater activity reduction than single mutations, with the degree of reduction correlating with the distance between mutation sites .

These findings suggest that proton translocation in the c-ring involves cooperative interaction between multiple subunits rather than independent functioning of each subunit. Experimental approaches to study these effects include:

• Site-directed mutagenesis targeting conserved residues

• Creation of genetically fused c-rings to control the number and position of mutations

• Measurement of ATP synthesis/hydrolysis activities

• Molecular dynamics simulations to visualize proton transfer events

What insights have been gained from molecular dynamics simulations of ATP synthase c-ring rotation?

Molecular dynamics (MD) simulations coupled with proton transfer algorithms have provided remarkable insights into the mechanism of c-ring rotation:

• Proton uptake and release dynamics: Simulations reveal that in wild-type ATP synthase, 2-3 deprotonated carboxyl residues typically face the a-subunit simultaneously .

• Cooperativity mechanisms: The waiting time for proton uptake can be shared between adjacent c-subunits, explaining the experimental observation that activity decreases as mutated subunits are placed farther apart .

• Distance-dependent effects: Simulations confirm that cooperative effects diminish when mutations are separated by more than three c-subunits in the ring, consistent with experimental findings .

The table below summarizes key findings from simulations of wild-type and mutant ATP synthases:

These findings collectively support a model where at least three c-subunits at the a/c interface cooperate during c-ring rotation .

How do environmental conditions affect the expression of atpH in Phaseolus vulgaris?

The expression of atpH in Phaseolus vulgaris is influenced by various environmental factors, particularly those affecting photosynthetic activity and energy demands. Under phosphorus deficiency, a common stress factor for bean production in many regions, plants must adjust their energy metabolism . Research indicates that:

• Phosphorus limitation affects the expression of genes involved in energy metabolism, including those encoding ATP synthase components.

• Genotypic differences exist in how bean varieties respond to phosphorus stress, with varieties like MILENIO, BAT477, and A785
  showing superior performance under low phosphorus conditions .

• Adaptation mechanisms may involve modifications in ATP synthase efficiency or expression levels to maintain energy production under limiting conditions.

Experimental approaches to study these effects include:

• RT-qPCR analysis of atpH expression under various phosphorus levels

• Proteomic analysis to quantify ATP synthase subunit abundance

• Measurement of ATP synthesis capacity in isolated chloroplasts from plants grown under different conditions

• Correlation of expression data with physiological parameters such as biomass production and phosphorus uptake

What strategies exist for enhancing the stability and activity of recombinant ATP synthase subunit c?

Enhancing the stability and activity of recombinant ATP synthase subunit c requires addressing several challenges inherent to membrane proteins:

• Optimization of expression constructs:

  • Use of strong, inducible promoters

  • Codon optimization for the expression host

  • Addition of purification tags that minimally impact function

  • Inclusion of appropriate signal sequences for membrane insertion

• Host system selection:

  • E. coli systems typically yield multiple forms of the protein (correctly processed, formyl-methionyl, and truncated versions)

  • Alternative expression hosts (yeast, insect cells) may provide better processing

  • Cell-free systems can be advantageous for toxic membrane proteins

• Purification and stabilization strategies:

  • Careful selection of detergents compatible with protein function

  • Use of lipid nanodiscs or amphipols to maintain native-like environment

  • Addition of stabilizing agents during purification

• Functional reconstitution:

  • Selection of appropriate lipid composition for proteoliposomes

  • Controlled protein-to-lipid ratios to ensure proper assembly

  • Addition of other ATP synthase subunits to stabilize the c-subunit structure

These strategies must be empirically optimized for the specific recombinant protein to balance yield, purity, stability, and biological activity.

What advanced structural techniques can be applied to study the c-ring assembly?

Advanced structural techniques provide critical insights into c-ring assembly and function:

• Cryo-electron microscopy (cryo-EM):

  • Enables visualization of the entire ATP synthase complex at near-atomic resolution

  • Can capture different conformational states during the rotational cycle

  • Requires minimal sample amounts compared to crystallography

• X-ray crystallography:

  • Has successfully resolved several c-ring structures at high resolution

  • Reveals precise arrangement of subunits and key residues

  • Requires large amounts of highly pure, homogeneous protein

• Solid-state NMR spectroscopy:

  • Provides atomic-level information about membrane proteins in a native-like environment

  • Can detect dynamic processes relevant to proton translocation

  • Requires isotopic labeling of the recombinant protein

• Cross-linking mass spectrometry:

  • Identifies interaction surfaces between c-subunits and with other ATP synthase components

  • Helps validate structural models and assembly intermediates

  • Can be combined with mutagenesis to map functional domains

• Molecular dynamics simulations:

  • Enables visualization of proton movement through the c-ring

  • Can model effects of mutations on structure and function

  • Provides insights into cooperative mechanisms between subunits

The combination of these techniques provides a comprehensive understanding of c-ring structure, assembly, and dynamic function beyond what any single method can achieve.

How can researchers troubleshoot low yields or activity in recombinant ATP synthase subunit c production?

Troubleshooting low yields or activity of recombinant ATP synthase subunit c requires systematic analysis of the expression and purification process:

• Expression troubleshooting:

  • Verify plasmid sequence integrity

  • Test multiple expression conditions (temperature, induction time, inducer concentration)

  • Consider alternative expression hosts or codon-optimized constructs

  • Implement toxic gene control strategies (tighter promoter control, secretion approaches)

• Extraction optimization:

  • Test different cell lysis methods (sonication, French press, detergent-based)

  • Optimize buffer conditions (pH, salt concentration, reducing agents)

  • Screen detergent types and concentrations for maximum extraction

  • Consider inclusion body refolding protocols if applicable

• Purification refinement:

  • Implement multi-step chromatography to separate different forms of the protein

  • Analyze each fraction for purity and specific activity

  • Remember that formyl-methionyl versions typically retain only ~10% activity

  • Monitor for truncated forms like those missing N-terminal residues (e.g., atpH-(8-84)) which may be inactive

• Activity analysis:

  • Compare activity to appropriate standards using multiple assay types

  • Ensure reconstitution conditions promote proper folding and assembly

  • Verify structural integrity through methods described in previous questions

  • Consider the impact of any purification tags on function

Systematic documentation of optimization attempts will help identify critical parameters affecting yield and activity.

What emerging technologies might advance our understanding of ATP synthase subunit c function?

Several emerging technologies show promise for advancing our understanding of ATP synthase subunit c function:

• Single-molecule techniques:

  • Single-molecule FRET to directly observe conformational changes during rotation

  • Optical trapping to measure the force generation during c-ring rotation

  • High-speed AFM to visualize rotation in real-time

• Integrative structural biology approaches:

  • Combining cryo-EM, X-ray crystallography, and NMR data for complete structural models

  • Time-resolved structural methods to capture transient states during the catalytic cycle

  • In-cell structural studies to examine ATP synthase in its native environment

• Advanced computational methods:

  • Quantum mechanics/molecular mechanics (QM/MM) simulations for proton transfer events

  • Machine learning approaches to predict functional impacts of mutations

  • Systems biology models integrating ATP synthase function with cellular energetics

• Synthetic biology strategies:

  • Designer c-rings with altered stoichiometry or properties

  • Integration of ATP synthase components into artificial photosynthetic systems

  • Development of minimal functioning ATP synthase models

These technologies will help address fundamental questions about the mechanisms of energy conversion and coupling between proton movement and ATP synthesis.

How might research on Phaseolus vulgaris ATP synthase contribute to understanding plant adaptation to environmental stresses?

Research on Phaseolus vulgaris ATP synthase has significant implications for understanding plant adaptation to environmental stresses:

• Phosphorus deficiency adaptation:

  • Different bean genotypes show varying abilities to maintain growth under phosphorus limitation

  • Superior varieties like MILENIO, BAT477, and A785 demonstrate enhanced root and shoot biomass production under low phosphorus conditions

  • Understanding how ATP synthase function is maintained under these conditions could reveal adaptation mechanisms

• Energy efficiency mechanisms:

  • Variations in ATP synthase subunit c might affect the H+/ATP ratio or coupling efficiency

  • More efficient ATP production could contribute to stress tolerance

  • Comparison between stress-resistant and sensitive varieties may reveal adaptive modifications

• Evolutionary adaptations:

  • Comparative studies across Phaseolus varieties adapted to different environments

  • Identification of natural variations in atpH that correlate with environmental adaptation

  • Assessment of selection pressure on ATP synthase components

• Applications in crop improvement:

  • Identification of superior alleles for breeding programs

  • Development of markers associated with energy efficiency traits

  • Potential targets for genetic engineering to enhance stress tolerance

This research connects fundamental bioenergetic mechanisms with practical applications in agriculture, particularly for regions where beans are cultivated under challenging conditions with limited inputs .