Recombinant Sorghum bicolor ATP synthase subunit c, chloroplastic (atpH)

What is the structure and function of ATP synthase subunit c, chloroplastic (atpH) in Sorghum bicolor?

ATP synthase subunit c, chloroplastic (atpH) in Sorghum bicolor is a small membrane protein (81 amino acids) that constitutes the primary component of the c-ring within the F0 sector of chloroplast ATP synthase. The amino acid sequence is: MNPLIAAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV .

The protein functions as part of a cylindrical oligomer (c-ring) that rotates during ATP synthesis. This rotation is coupled to proton translocation across the thylakoid membrane, which drives the synthesis of ATP in the F1 portion of the enzyme. The c-ring serves as the rotor in this molecular motor, converting the proton gradient energy into mechanical rotation energy that drives ATP synthesis .

This subunit c is also known by several alternative names:

• ATP synthase F(0) sector subunit c

• ATPase subunit III

• F-type ATPase subunit c

• F-ATPase subunit c

• Lipid-binding protein

How does recombinant atpH protein differ from native protein in structure and function?

Recombinant Sorghum bicolor atpH produced in expression systems maintains the same primary amino acid sequence as the native protein but may exhibit differences in post-translational modifications and folding characteristics. When expressed in heterologous systems, recombinant atpH typically requires optimized buffer conditions (Tris-based buffer with 50% glycerol) to maintain stability and proper conformation .

Unlike native atpH isolated from Sorghum bicolor chloroplasts, recombinant versions often include affinity tags for purification purposes. These tags are generally determined during the production process and may affect certain biophysical properties while enabling easier purification . In functional studies, recombinant atpH must be properly refolded and incorporated into membrane environments to recapitulate native activity, as its hydrophobic nature requires specific handling protocols to prevent aggregation.

For structural integrity validation, circular dichroism spectroscopy can be used to confirm that recombinant atpH maintains the correct alpha-helical secondary structure characteristic of the native protein . Functional reconstitution studies have demonstrated that properly produced recombinant subunit c can be assembled into functional c-rings with properties similar to those of native protein complexes.

What expression systems are most effective for producing recombinant Sorghum bicolor atpH?

Several expression systems have been evaluated for the production of recombinant ATP synthase subunit c, with varying levels of success:

Bacterial Expression Systems: Escherichia coli has been successfully used to express recombinant chloroplast ATP synthase subunit c, particularly when fused to solubility-enhancing partners such as maltose-binding protein (MBP). This approach has yielded milligram quantities of purified protein. Key optimization parameters include:

• Use of low-copy number expression vectors

• Induction at lower temperatures (16-20°C)

• Expression in E. coli strains optimized for membrane proteins (C41/C43)

Yeast Expression Systems: Pichia pastoris has emerged as an efficient eukaryotic host for expressing plant membrane proteins, including ATP synthase components. This system offers advantages for plant proteins that require specific post-translational modifications .

Cell-Free Systems: For difficult-to-express membrane proteins like atpH, cell-free expression systems have been employed with supplementation of lipids or detergents to improve protein solubility and folding.

To optimize expression, researchers should consider:

• Codon optimization for the host organism

• Signal peptide modifications for appropriate targeting

• Growth temperature and induction conditions

• Selection of appropriate detergents for extraction and purification

• Buffer composition during purification steps

What purification strategies are most effective for recombinant Sorghum bicolor atpH?

Purifying recombinant atpH requires specialized approaches due to its hydrophobic nature. The most effective purification strategies include:

Affinity Chromatography:

• Histidine-tagged constructs: Purification using Ni-NTA resin with optimized imidazole gradients

• MBP-fusion proteins: Amylose resin purification followed by tag cleavage using specific proteases

• Both approaches require detergent incorporation throughout purification steps

Size Exclusion Chromatography:

• Critical for separating monomeric atpH from aggregated forms

• Typically performed in buffers containing 0.05-0.1% mild detergents (DDM, LDAO)

Specialized Buffer Components:
For maximum stability during purification, the following buffer components have proven most effective:

• Base buffer: 50 mM Tris (pH 6.8-7.5)

• Stabilizers: 50% glycerol

• Protease inhibitors: 1 mM phenylmethylsulfonyl fluoride

• Chelators: 0.1 mM EDTA

Storage Conditions:

• Store at -20°C for short-term or -80°C for extended storage

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

This purification protocol has been validated to produce highly purified protein suitable for structural and functional studies of atpH.

What methods are most reliable for validating the structure and function of recombinant atpH?

Validating recombinant atpH requires multiple complementary approaches to confirm both structural integrity and functional activity:

Structural Validation:

• Circular Dichroism (CD) Spectroscopy: Confirms alpha-helical secondary structure characteristic of ATP synthase subunit c

• Mass Spectrometry: Verifies primary sequence and post-translational modifications

• NMR Spectroscopy: Provides detailed structural information in membrane-mimetic environments

• Cryo-EM: When assembled into c-rings, validates oligomeric structure

Functional Validation:

• Reconstitution into liposomes to measure proton translocation

• DCCD (dicyclohexylcarbodiimide) binding assays: DCCD specifically binds to the functional proton-binding site on subunit c

  • pH-dependent DCCD labeling profiles can determine the proton-binding characteristics

  • Chloroplast ATP synthases typically exhibit bell-shaped pH profiles for DCCD labeling

• Assembly assays to verify incorporation into larger ATP synthase complexes

Table 1: DCCD Labeling Profiles for Different ATP Synthase Types

This comparative data helps validate the functional properties of recombinant atpH against known patterns .

How can recombinant atpH be used to study c-ring stoichiometry variation across species?

The c-ring stoichiometry (number of c subunits per ring) varies among organisms (ranging from c10 to c15) and directly affects the bioenergetic efficiency of ATP synthesis. Recombinant atpH provides a powerful tool for investigating this phenomenon:

Methodological Approach:

• Express and purify recombinant atpH from Sorghum bicolor

• Reconstitute c-rings in vitro under controlled conditions

• Determine stoichiometry using techniques such as:

  • Mass photometry

  • Atomic force microscopy

  • Native mass spectrometry

  • Cryo-electron microscopy

Research Applications:

• Comparing wild-type and mutant atpH variants to identify determinants of c-ring assembly

• Investigating the relationship between c-ring size and ATP synthesis efficiency

• Creating chimeric c-subunits to identify regions responsible for species-specific stoichiometry

The c-ring stoichiometry directly determines the H+/ATP ratio during ATP synthesis. For example, a c14 ring results in a ratio of 4.7 H+/ATP, while a c10 ring yields a ratio of 3.3 H+/ATP. This variation has significant implications for bioenergetic efficiency across different photosynthetic organisms .

Table 2: Relationship Between c-Ring Stoichiometry and Coupling Ratio

This research direction is particularly valuable for understanding the evolutionary adaptation of photosynthetic efficiency in different plant species, including Sorghum bicolor .

What role does atpH play in chloroplast redox regulation in Sorghum bicolor?

Chloroplast ATP synthase (CFoCF1) is unique among ATP synthase complexes due to its redox regulation mechanism, which is essential for balancing ATP production with fluctuating light conditions during photosynthesis:

Key Research Findings:

• While the redox-sensitive cysteines are located on the γ subunit rather than the c subunit (atpH), the c-ring's rotation directly influences the conformational changes required for redox regulation

• The c-ring of CFoCF1 interacts with both the γ and ε subunits, creating a central stalk that transmits rotation to the catalytic sites

• Under oxidizing (dark) conditions, disulfide bonds form in the γ subunit, affecting interactions with the rotating c-ring

Experimental Approaches to Study This Relationship:

• Site-directed mutagenesis of specific atpH residues that interact with redox-regulated subunits

• Reconstitution of ATP synthase complexes with wild-type or modified atpH

• Measurement of ATP synthesis rates under varying redox conditions

• Analysis of reactive oxygen species (ROS) production in relation to ATP synthase activity

Table 3: Redox Regulation Effects on ATP Synthase Activity

Understanding this relationship is particularly important in crop plants like Sorghum bicolor, where efficient energy conversion under varying light conditions directly impacts agricultural productivity .

How can recombinant atpH be used to study cytoplasmic male sterility in Sorghum bicolor?

Cytoplasmic male sterility (CMS) in Sorghum bicolor has been linked to mitochondrial ATP synthase genes, with evidence suggesting that ATP synthase subunits may be implicated in this important agricultural trait:

Research Evidence:

• Polymorphisms in ATP synthase genes have been observed between male sterile (A) and maintainer fertile (B) lines of Sorghum bicolor

• Transcript size differences in ATP synthase genes (particularly atpA) exist between A and B cytoplasms

• These differences may lead to incompatible subunits being synthesized by mitochondria and nucleus for the ATP synthase complex

Experimental Approaches Using Recombinant atpH:

• Compare the sequences of atpH from sterile and fertile Sorghum bicolor lines

• Express recombinant versions of both variants

• Perform interaction studies with other ATP synthase subunits

• Reconstitute hybrid ATP synthase complexes with mixed subunit origins

• Analyze ROS production and ATP synthesis efficiency in reconstituted systems

Studies in cotton have demonstrated that ATP synthase subunit genes (atpE and atpF) are closely linked with ROS metabolism and programmed cell death in anthers, which are key processes in male sterility. Similar mechanisms may operate in Sorghum bicolor .

Table 4: Comparison of ATP Synthase Gene Expression in Male Sterile vs. Fertile Lines

This research direction has significant implications for hybrid seed production in Sorghum bicolor and other important crops .

How can protein aggregation be prevented during recombinant atpH purification?

Protein aggregation is a common challenge when working with highly hydrophobic membrane proteins like atpH. Several strategies have proven effective in preventing aggregation:

Optimized Solubilization Conditions:

• Detergent selection: Mild detergents like DDM (n-dodecyl β-D-maltoside), LDAO, or Brij-35 at concentrations just above their critical micelle concentration

• Lipid supplementation: Addition of phospholipids (0.1-0.5 mg/ml) to stabilize the protein

• Proper protein:detergent ratios are critical for maintaining solubility

Buffer Optimization:

• Include stabilizing agents: 50% glycerol has been demonstrated to effectively stabilize atpH

• Salt concentration: 150-300 mM NaCl helps prevent ionic interactions leading to aggregation

• pH optimization: Maintain pH between 6.8-7.5 for maximum stability

Expression Strategies:

• Fusion partners: Expression as MBP-fusion proteins significantly improves solubility

• Low temperature expression: Induction at 16-20°C reduces inclusion body formation

• Codon optimization: Enhances translation efficiency and proper folding

Handling Protocols:

• Avoid concentration above 5 mg/ml, which often triggers aggregation

• Store in smaller aliquots to minimize freeze-thaw cycles

• Keep samples at 4°C rather than room temperature when possible

Implementing these approaches has been shown to reduce aggregation by >80% during purification of recombinant ATP synthase subunits, resulting in higher yields of functional protein.

How can researchers troubleshoot non-functional recombinant atpH in reconstitution experiments?

When recombinant atpH fails to function properly in reconstitution experiments, several methodological approaches can help identify and resolve the issues:

Systematic Troubleshooting Protocol:

• Verify Protein Integrity:

  • Confirm correct molecular weight by SDS-PAGE and mass spectrometry

  • Validate secondary structure using circular dichroism spectroscopy

  • Assess oligomeric state using size exclusion chromatography

• Optimize Reconstitution Conditions:

  • Test multiple lipid compositions (varying MGDG:DGDG ratios for chloroplast membrane mimics)

  • Optimize protein-to-lipid ratios (typically 1:50 to 1:200 w/w)

  • Evaluate different reconstitution methods (dialysis vs. dilution vs. direct incorporation)

• Validate Membrane Insertion:

  • Perform protease protection assays to confirm proper membrane integration

  • Use fluorescence quenching to assess protein orientation in membranes

  • Apply freeze-fracture electron microscopy to visualize protein distribution

• Assess Functional Parameters:

  • Test DCCD binding at various pH values (pH 5-11) to verify proton-binding site integrity

  • Measure proton translocation using pH-sensitive fluorescent dyes

  • Evaluate interaction with other ATP synthase subunits using pull-down assays

Common Issues and Solutions:

Implementing this systematic approach can significantly improve the success rate of functional reconstitution experiments with recombinant atpH .

How might genetic engineering of atpH be used to enhance photosynthetic efficiency in Sorghum bicolor?

Genetic engineering of atpH represents a promising frontier for enhancing photosynthetic efficiency in Sorghum bicolor, with several strategic approaches:

Targeted Modification Strategies:

• c-Ring Stoichiometry Engineering: Altering the number of c subunits per ring could optimize the H+/ATP ratio, potentially improving energy conversion efficiency under specific environmental conditions

• Proton-Binding Site Modifications: Strategic mutations in key residues could alter proton affinity, potentially accelerating ATP synthesis rates

• Interface Optimization: Modifying interfaces between atpH and other ATP synthase subunits could enhance complex stability and assembly efficiency

Experimental Approaches:

• Design atpH variants with modified sequence characteristics

• Express and characterize recombinant proteins to validate in vitro properties

• Transform Sorghum bicolor using plastid transformation techniques

• Assess photosynthetic parameters, including:

  • ATP synthesis rates

  • Electron transport rates

  • CO2 assimilation efficiency

  • Growth under varying light conditions

Potential Benefits:

• Improved drought tolerance through more efficient energy usage

• Enhanced biomass production under suboptimal conditions

• Better adaptation to fluctuating light environments

This research direction builds on understanding the unique properties of chloroplast ATP synthase, particularly its redox regulation mechanism that is critical for balancing energy production with environmental conditions .

What novel approaches can be used to study interactions between atpH and other ATP synthase subunits?

Advanced methodologies for studying protein-protein interactions provide new opportunities to understand the complex assembly and function of ATP synthase components:

Cutting-Edge Methodological Approaches:

• Nanobody Discovery and Application:

  • mRNA/cDNA display technology can be used to discover native conformation-binding nanobodies against purified atpH

  • These nanobodies can serve as specific molecular probes to study interactions and conformational changes

  • The technique is particularly valuable as it allows animal-free antibody generation against non-immunogenic plant proteins

• Cryo-EM of Partially Assembled Complexes:

  • Expression of atpH with select partner subunits

  • Visualization of assembly intermediates via cryo-EM

  • Determination of specific interaction interfaces

• In situ Labeling Techniques:

  • Genetic code expansion to incorporate photo-crosslinkable amino acids at specific sites

  • UV-induced crosslinking to capture transient interactions

  • Mass spectrometry analysis of crosslinked peptides to map interaction sites

• Förster Resonance Energy Transfer (FRET) Approaches:

  • Site-specific labeling of atpH and partner subunits with fluorophore pairs

  • Real-time monitoring of protein-protein interactions

  • Detection of conformational changes during enzymatic cycles

Research Applications:

• Identifying critical residues for c-ring assembly and stability

• Mapping the interaction surfaces between atpH and subunits of the central stalk

• Understanding the molecular basis for species-specific differences in ATP synthase assembly

These approaches extend beyond traditional co-immunoprecipitation and yeast two-hybrid methods, providing more detailed spatial and temporal information about protein interactions within the ATP synthase complex .

What is the potential role of atpH in bioenergy applications beyond natural photosynthesis?

ATP synthase subunit c (atpH) has unique properties that could be exploited in various bioenergy applications, representing an exciting frontier in sustainable energy research:

Innovative Bioenergy Applications:

• Biohybrid Energy Conversion Systems:

  • Integration of recombinant c-rings into artificial membranes

  • Creation of proton gradient-driven ATP production systems

  • Coupling with light-harvesting nanoparticles for artificial photosynthesis

• Bioelectric Interfaces:

  • Development of c-ring-based proton conduction channels

  • Integration with electrodes for bioelectricity generation

  • Creation of bio-inspired fuel cells using proton gradients

• Biomimetic Nanomotors:

  • Engineering of synthetic rotary motors based on c-ring structure

  • Creation of nanoscale devices powered by proton gradients

  • Development of ATP-producing nanomachines

Research Approaches:

• Structure-guided design of modified c-rings with enhanced stability

• Reconstitution into various synthetic membrane systems

• Coupling with artificial electron transport chains

• Integration with non-biological energy harvesting systems

The exceptional efficiency of the c-ring as a biological rotary motor (nearly 100% efficiency in converting proton gradient energy to mechanical rotation) makes it an ideal component for developing bio-inspired energy conversion technologies. By understanding and manipulating the structure-function relationship of atpH from photosynthetic organisms like Sorghum bicolor, researchers can potentially develop novel bioenergy solutions that exceed the efficiency of current technologies .