Recombinant Ipomoea purpurea ATP synthase subunit c, chloroplastic (atpH)

What is ATP synthase subunit c in Ipomoea purpurea and what is its role in chloroplast function?

ATP synthase subunit c (atpH) in Ipomoea purpurea is a small, hydrophobic protein component of the chloroplastic ATP synthase complex. This protein forms part of the membrane-embedded c-ring within the FO portion of the ATP synthase. The c-ring plays a critical role in proton translocation across the thylakoid membrane, which drives the synthesis of ATP through the rotary mechanism of the ATP synthase complex. In chloroplasts, this process is essential for converting the energy captured during photosynthesis into chemical energy stored in ATP molecules, which then power various metabolic processes in the plant cell .

The subunit c is characterized by its alpha-helical secondary structure, which is crucial for its proper assembly into the c-ring oligomer. This structural feature is conserved across different species, including the well-studied spinach chloroplast ATP synthase subunit c . The protein's small size (typically around 8 kDa) and highly hydrophobic nature present significant challenges for recombinant production and purification, necessitating specialized approaches similar to those developed for other species.

What techniques are most reliable for initial characterization of recombinant atpH from Ipomoea purpurea?

The most reliable techniques for initial characterization of recombinant atpH include:

• SDS-PAGE with tricine buffer systems: This provides effective separation of low molecular weight proteins like subunit c.

• Western blotting: Using antibodies specific to conserved regions of subunit c helps confirm the identity of the recombinant protein.

• Circular dichroism (CD) spectroscopy: This technique is essential for confirming the correct alpha-helical secondary structure of the purified recombinant protein, as demonstrated with spinach chloroplast ATP synthase subunit c .

• Mass spectrometry: This provides precise molecular weight determination and can confirm post-translational modifications.

• Amino acid composition analysis: This verifies the correct primary structure of the recombinant protein.

For functional characterization, reconstitution of the purified subunit c into liposomes followed by proton translocation assays can provide insights into its functional integrity. Researchers should also consider comparative analyses with well-characterized ATP synthase subunit c from other plant species like spinach to validate their findings .

How does the atpH gene structure and regulation differ between Ipomoea purpurea and other plant models?

The expression of chloroplast genes, including atpH, is often regulated by light-dependent mechanisms and developmental cues. In Ipomoea purpurea, which belongs to the Convolvulaceae family, regulatory elements may have evolved specific adaptations related to its growth habit and environmental responses . Transcription factors such as bHLH proteins, which are known to regulate various biosynthetic pathways in Ipomoea purpurea , might also play indirect roles in modulating energy metabolism genes including those involved in ATP synthesis.

A comprehensive analysis of atpH gene structure in Ipomoea purpurea would require genome sequence comparison with other plant species, identification of conserved regulatory elements, and experimental validation of promoter activities under various conditions.

What are the optimal expression systems and conditions for producing functional recombinant Ipomoea purpurea ATP synthase subunit c?

Based on successful approaches with other chloroplastic ATP synthase subunit c proteins, the following expression systems and conditions are recommended for Ipomoea purpurea atpH:

Expression Systems Comparison:

For optimal results with the E. coli system, the use of fusion partners like maltose-binding protein (MBP) has proven effective for ATP synthase subunit c from spinach chloroplasts . The expression protocol should include:

• Growth at lower temperatures (16-20°C) after induction

• Reduced IPTG concentration (0.4-0.5 mM)

• Extended expression time (18-24 hours)

• Rich media supplemented with glucose to suppress basal expression

• Inclusion of membrane-stabilizing agents in the culture medium

These conditions help minimize the formation of inclusion bodies while maintaining the proper folding of the hydrophobic subunit c protein. After expression, a two-step purification strategy involving affinity chromatography followed by size exclusion chromatography typically yields the highest purity protein suitable for structural and functional studies .

How can researchers effectively assess the assembly and oligomerization properties of recombinant Ipomoea purpurea atpH?

Assessing the assembly and oligomerization properties of recombinant Ipomoea purpurea atpH requires specialized techniques suitable for membrane proteins:

• Blue Native PAGE (BN-PAGE): This technique allows visualization of intact protein complexes under non-denaturing conditions. By comparing migration patterns with established standards, researchers can determine the oligomeric state of the reconstituted c-ring.

• Analytical Ultracentrifugation: Sedimentation velocity and equilibrium experiments provide valuable information about the molecular weight and homogeneity of protein complexes in solution.

• Chemical Cross-linking followed by Mass Spectrometry: This approach can capture transient interactions and identify specific residues involved in subunit-subunit contacts within the c-ring.

• Electron Microscopy and Single-particle Analysis: These techniques allow direct visualization of c-ring structure and determination of the number of c-subunits per ring.

• Förster Resonance Energy Transfer (FRET): By introducing fluorescent labels at strategic positions, researchers can monitor proximity relationships and conformational changes during assembly.

A critical consideration is the lipid environment, as the composition of the membrane can significantly influence assembly properties. Researchers should systematically test different lipid compositions that mimic the native thylakoid membrane of Ipomoea purpurea chloroplasts. Additionally, the presence of other ATP synthase components (particularly subunits a and b) may be necessary to facilitate proper assembly and stability of the c-ring structure.

What are the functional consequences of sequence variations in the atpH gene across different Ipomoea species?

Sequence variations in the atpH gene across different Ipomoea species can have significant functional consequences for ATP synthase activity and plant energy metabolism. While the core functional regions of subunit c are highly conserved due to their essential role in proton translocation, species-specific variations may fine-tune the protein's properties in response to different ecological niches and environmental adaptations.

Potential functional consequences include:

Comparative studies using recombinant atpH proteins from different Ipomoea species, combined with site-directed mutagenesis of specific variable residues, would provide valuable insights into the functional significance of these sequence differences. Such information could enhance our understanding of evolutionary adaptations in energy metabolism across the Convolvulaceae family.

What purification strategies yield the highest purity and activity for recombinant Ipomoea purpurea ATP synthase subunit c?

Purification of recombinant Ipomoea purpurea ATP synthase subunit c requires specialized approaches due to its hydrophobic nature. The following multi-step strategy has been demonstrated to be effective for similar proteins:

Recommended Purification Protocol:

• Membrane Fraction Isolation:

  • Cell lysis using French press or sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl₂, 10% glycerol

  • Differential centrifugation to isolate membrane fractions (40,000 × g for 1 hour)

  • Membrane solubilization using detergents (1-2% n-dodecyl-β-D-maltoside or 1% Triton X-100)

• Affinity Chromatography:

  • For MBP fusion constructs, amylose resin affinity chromatography

  • Thorough washing with detergent-containing buffer to remove contaminants

  • Elution with 10 mM maltose for MBP-tagged proteins

• Tag Removal:

  • Proteolytic cleavage using Factor Xa or PreScission protease

  • Incubation at 4°C for 16-18 hours in optimized buffer conditions

• Secondary Purification:

  • Size exclusion chromatography using Superdex 75 or 200 columns

  • Ion exchange chromatography with carefully optimized pH and salt gradients

• Final Polishing:

  • Hydroxyapatite chromatography to remove residual contaminants

  • Concentration using centrifugal filters with appropriate molecular weight cutoffs

Throughout the purification process, it is critical to maintain the presence of appropriate detergents or lipids to prevent aggregation of the hydrophobic subunit c. The purification buffers should also contain stabilizing agents such as glycerol (10-15%) and low concentrations of reducing agents like DTT or β-mercaptoethanol to prevent oxidation of susceptible residues.

The purity of the final preparation can be assessed using silver-stained SDS-PAGE, while the structural integrity can be confirmed by circular dichroism spectroscopy to verify the characteristic alpha-helical secondary structure, as demonstrated with spinach chloroplast ATP synthase subunit c .

How can researchers optimize codon usage for enhanced expression of Ipomoea purpurea atpH in heterologous systems?

Codon optimization is crucial for enhancing the expression of Ipomoea purpurea atpH in heterologous systems, particularly in bacterial hosts like E. coli. The following methodological approach is recommended:

Codon Optimization Workflow:

• Analysis of Native Sequence:

  • Calculate the Codon Adaptation Index (CAI) of the native atpH sequence

  • Identify rare codons that might cause translational pausing or premature termination

  • Map the GC content distribution, which can affect mRNA secondary structure

• Host-Specific Optimization:

  • Replace rare codons with synonymous codons preferred by the expression host

  • For E. coli expression, consider using the following codon preference substitutions:

    • AUA (Ile) → AUC

    • CUA (Leu) → CUG

    • AGA/AGG (Arg) → CGU/CGC

    • CGA (Arg) → CGU/CGC

    • GGA (Gly) → GGU/GGC

• mRNA Secondary Structure Optimization:

  • Modify the 5' region to reduce stable secondary structures

  • Eliminate potential ribosome binding sites within the coding sequence

  • Optimize the translation initiation region for efficient ribosome binding

• Removal of Problematic Sequence Elements:

  • Eliminate internal Shine-Dalgarno-like sequences

  • Remove sequences that might act as cryptic splice sites in eukaryotic hosts

  • Avoid sequences that create mRNA instability elements

• Experimental Validation:

  • Compare expression levels between native and optimized sequences

  • Fine-tune optimization based on experimental results

For Ipomoea purpurea atpH, a comparison of different optimization algorithms (GeneArt, GeneOptimizer, JCat) can identify the most effective approach. Additionally, researchers should consider using specialized E. coli strains like Rosetta or BL21-CodonPlus that supply additional tRNAs for rare codons if complete codon optimization is not feasible.

Preliminary studies with spinach chloroplast ATP synthase subunit c demonstrated that fusion to MBP significantly enhanced expression compared to other fusion partners , suggesting that optimization of the expression construct design is as important as codon optimization.

What techniques enable accurate structural characterization of recombinant Ipomoea purpurea ATP synthase subunit c?

Accurate structural characterization of recombinant Ipomoea purpurea ATP synthase subunit c requires a multi-faceted approach combining various biophysical techniques:

Structural Characterization Techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Provides information about secondary structure composition

  • Confirms the expected alpha-helical content (typically >70% for subunit c)

  • Allows monitoring of structural stability under different conditions (pH, temperature, detergents)

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Provides atomic-level structural information in solution

  • Particularly valuable for membrane proteins when combined with detergent micelles or nanodiscs

  • Requires isotopic labeling (¹⁵N, ¹³C) of the recombinant protein

• X-ray Crystallography:

  • Yields high-resolution structural information

  • Requires successful crystallization of the protein (challenging for membrane proteins)

  • May require lipidic cubic phase (LCP) crystallization methods

• Cryo-Electron Microscopy (Cryo-EM):

  • Increasingly powerful for membrane protein structure determination

  • Can visualize the entire ATP synthase complex or isolated c-ring

  • Does not require crystallization

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Provides information about protein dynamics and solvent accessibility

  • Identifies regions involved in protein-protein or protein-lipid interactions

  • Complementary to static structural methods

For integrated structural analysis, researchers should systematically characterize the recombinant protein in different environments, including detergent micelles, nanodiscs, and reconstituted liposomes. Comparative analysis with known structures of ATP synthase subunit c from other species can provide valuable insights into conserved structural features and species-specific variations.

The structural integrity of recombinant subunit c can be further validated using functional assays that measure proton translocation activity after reconstitution into liposomes, ensuring that the recombinant protein not only has the correct structure but also retains its native function.

How can recombinant Ipomoea purpurea ATP synthase subunit c be used to study energy coupling mechanisms in photosynthesis?

Recombinant Ipomoea purpurea ATP synthase subunit c serves as a valuable tool for investigating energy coupling mechanisms in photosynthesis through several experimental approaches:

• Reconstitution Studies:

  • Recombinant subunit c can be reconstituted with other ATP synthase components to form functional complexes

  • These reconstituted systems allow precise manipulation of component stoichiometry and introduction of specific mutations

  • Researchers can measure ATP synthesis rates under controlled proton gradient conditions

• Proton Translocation Assays:

  • Purified recombinant subunit c can be incorporated into liposomes containing pH-sensitive fluorescent dyes

  • This system enables direct measurement of proton translocation efficiency and kinetics

  • Comparison of wild-type and mutant forms provides insights into structure-function relationships

• Protein-Protein Interaction Studies:

  • Using techniques like surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

  • These methods quantify binding affinities between subunit c and other components of the ATP synthase

  • Identifying critical interaction surfaces that mediate complex assembly and function

• Inhibitor Binding Studies:

  • Recombinant subunit c can be used to characterize interactions with known ATP synthase inhibitors

  • This approach helps identify species-specific differences in inhibitor sensitivity

  • Results contribute to understanding the molecular mechanisms of ATP synthase inhibition

By combining these approaches, researchers can develop detailed models of energy coupling in Ipomoea purpurea chloroplasts, potentially revealing adaptations specific to this plant species. Such studies are particularly valuable when compared with well-characterized systems like spinach chloroplast ATP synthase , highlighting both conserved mechanisms and species-specific variations in photosynthetic energy transduction.

What insights can comparative studies between wild-type and mutant forms of recombinant atpH provide about c-ring assembly and function?

Comparative studies between wild-type and mutant forms of recombinant atpH offer critical insights into c-ring assembly and function through systematic structure-function analysis:

Key Experimental Approaches:

• Site-Directed Mutagenesis Studies:

  • Mutation of the conserved proton-binding glutamate residue (typically Glu61 in plants)

  • Alterations to residues at the subunit-subunit interface

  • Modifications to lipid-interacting residues in the transmembrane regions

• Impact on Assembly:

  • Mutations affecting c-ring formation can be identified by altered migration patterns in BN-PAGE

  • Electron microscopy of reconstituted c-rings can reveal structural abnormalities

  • Cross-linking studies can identify altered subunit-subunit interactions

• Functional Consequences:

  • Measurement of proton translocation efficiency in reconstituted proteoliposomes

  • ATP synthesis assays when combined with other ATP synthase components

  • Determination of proton binding affinity using pH-dependent spectroscopic methods

Representative Results Table:

These comparative studies can reveal how specific amino acid positions contribute to the unique properties of Ipomoea purpurea ATP synthase, potentially identifying adaptations related to its ecological niche. The alpha-helical secondary structure known to be critical in ATP synthase subunit c can be systematically analyzed through mutations that disrupt helix formation or packing, providing insights into the structural basis of c-ring stability and function.

How can researchers effectively integrate recombinant Ipomoea purpurea atpH into biophysical studies of rotary catalysis?

Integrating recombinant Ipomoea purpurea atpH into biophysical studies of rotary catalysis requires specialized approaches to visualize and quantify the dynamic behavior of this molecular machine:

Methodological Framework:

• Fluorescence-Based Rotation Assays:

  • Site-specific labeling of recombinant atpH with fluorescent probes

  • Attachment of the reconstituted c-ring to a surface while allowing rotation

  • Real-time monitoring of rotation using fluorescence microscopy techniques

  • Analysis of step size, rotation speed, and pause durations

• Single-Molecule FRET Studies:

  • Strategic placement of FRET donor-acceptor pairs between c-subunits or between c-ring and stator components

  • Detection of conformational changes during the catalytic cycle

  • Correlation of FRET signals with ATP synthesis or hydrolysis events

• Magnetic Bead Rotation Assays:

  • Attachment of magnetic beads to the c-ring component

  • Application of controlled magnetic fields to drive or resist rotation

  • Measurement of torque generation and mechanical work

• High-Speed Atomic Force Microscopy (HS-AFM):

  • Direct visualization of conformational changes during rotation

  • Real-time monitoring of subunit rearrangements at nanometer resolution

  • Correlation of structural changes with functional states

Critical Considerations for Implementation:

• Site-Specific Labeling Strategies:

  • Introduction of unique reactive groups (cysteines, unnatural amino acids) at strategic positions

  • Verification that labels do not disrupt function using control biochemical assays

  • Optimization of labeling efficiency and specificity

• Surface Immobilization Approaches:

  • Development of oriented attachment methods that preserve rotational freedom

  • Use of PEG-based linkers to minimize non-specific interactions

  • Validation of activity after immobilization

• Data Analysis Frameworks:

  • Statistical methods for step detection and dwell time analysis

  • Correlation of rotation events with ATP hydrolysis or synthesis

  • Stochastic modeling of the rotary mechanism

By implementing these approaches with recombinant Ipomoea purpurea atpH, researchers can gain species-specific insights into the rotary catalysis mechanism, potentially revealing adaptations that optimize ATP synthesis efficiency in this plant species. Integration with structural information about the alpha-helical conformation of subunit c can provide a comprehensive understanding of how structure facilitates this remarkable molecular motion.

What are the most promising areas for future research on recombinant Ipomoea purpurea ATP synthase subunit c?

The most promising areas for future research on recombinant Ipomoea purpurea ATP synthase subunit c include:

These research directions will benefit from continued methodological advances in recombinant protein production and characterization, building upon established approaches for ATP synthase subunit c expression and purification . Interdisciplinary approaches combining structural biology, biophysics, and plant physiology will be particularly valuable for developing a comprehensive understanding of this critical component of photosynthetic energy conversion in Ipomoea purpurea.

How might research on Ipomoea purpurea ATP synthase contribute to broader understanding of plant bioenergetics?

Research on Ipomoea purpurea ATP synthase subunit c has the potential to make significant contributions to our broader understanding of plant bioenergetics in several key areas:

• Evolutionary Adaptations in Energy Metabolism:

  • Ipomoea purpurea represents an important plant family (Convolvulaceae) with distinct evolutionary history

  • Comparative studies can reveal how ATP synthase has evolved in response to different ecological pressures

  • Identification of convergent or divergent adaptations across plant lineages

• Structure-Function Relationships in Membrane Protein Complexes:

  • Analysis of the relationship between c-ring structure and functional properties

  • Insights into how specific amino acid changes influence proton translocation efficiency

  • Understanding of how membrane protein complexes achieve long-term stability in the dynamic thylakoid environment

• Integration of Chloroplast and Nuclear Genomes:

  • Investigation of how chloroplast-encoded components like atpH coordinate with nuclear-encoded ATP synthase subunits

  • Mechanisms of stoichiometric regulation ensuring proper complex assembly

  • Coevolution of interacting components encoded in different cellular compartments

• Energetic Optimization in Photosynthesis:

  • Understanding of how c-ring stoichiometry influences the H⁺/ATP ratio

  • Analysis of how this ratio is optimized for different photosynthetic strategies

  • Insights into the balance between energetic efficiency and cellular ATP demands

• Adaptation to Environmental Stresses:

  • Elucidation of how ATP synthase function is maintained under various stress conditions

  • Identification of regulatory mechanisms that modify ATP synthase activity

  • Development of strategies to enhance stress resilience through bioenergetic optimization