Recombinant Chloranthus spicatus ATP synthase subunit c, chloroplastic (atpH)

What is chloroplastic ATP synthase subunit c and what role does it play in photosynthesis?

ATP synthase subunit c (also called subunit III) is a critical component of the multimeric chloroplast ATP synthase complex that produces adenosine triphosphate (ATP) essential for photosynthetic metabolism. This hydrophobic protein forms a ring structure embedded in the thylakoid membrane, creating a rotary motor that couples proton translocation to ATP synthesis. The c-subunit ring physically rotates as protons move across the membrane along an electrochemical gradient, mechanically driving the synthesis of ATP in the catalytic region of the enzyme .

The subunit c ring connects the membrane-embedded F₀ portion to the catalytic F₁ portion through a γ-stalk. As the c-ring rotates, it drives the rotation of the γ-subunit within the α₃β₃ head structure where ATP synthesis occurs at the interface of α and β subunits. This mechanical coupling is fundamental to energy conversion in photosynthetic organisms .

How does the structure of ATP synthase subunit c contribute to its function?

The ATP synthase subunit c has a distinctive alpha-helical secondary structure that is crucial for its proper function. This structural characteristic allows the protein to be properly embedded in the thylakoid membrane and to form the cylindrical c-ring necessary for proton translocation. Research with recombinant subunit c has confirmed the presence of the correct alpha-helical secondary structure in purified proteins, validating that the recombinant versions maintain the native structural properties required for function .

The c-subunit contains specific amino acid residues that facilitate proton binding and release during the rotational catalysis process. These conserved residues are positioned to allow sequential proton binding and release as the ring rotates, directly coupling proton movement across the membrane to the mechanical rotation that drives ATP synthesis .

What expression systems are most effective for producing ATP synthase subunit c?

Multiple expression systems have been developed for producing recombinant ATP synthase subunit c, with varying levels of success depending on the specific research goals. The search results indicate several options for expression hosts:

The most well-documented system uses E. coli BL21 derivative cells with a codon-optimized gene insert. The hydrophobic c₁ subunit is expressed as a soluble MBP-c₁ fusion protein, then cleaved from the maltose binding protein (MBP) and purified using reversed phase column chromatography. This approach enables the soluble expression of an otherwise highly hydrophobic membrane protein .

How can the hydrophobic nature of subunit c be overcome in recombinant expression?

The extreme hydrophobicity of ATP synthase subunit c presents a significant challenge for recombinant expression. Researchers have developed several strategies to address this issue:

• Fusion protein approach: Expressing the c-subunit as a fusion with a highly soluble partner protein, such as maltose binding protein (MBP), significantly increases solubility. This method has been successfully implemented for spinach chloroplast ATP synthase subunit c, with the MBP-c₁ fusion successfully expressed in soluble form .

• Codon optimization: Synthetic gene design with codons optimized for the expression host (e.g., E. coli) improves translation efficiency. For chloroplast ATP synthase subunit c, researchers have used Gene Designer software to create synthetic genes with E. coli-optimized codons and added terminal restriction sites for cloning .

• Chaperone co-expression: Co-transformation with plasmids expressing chaperone proteins such as DnaK, DnaJ, and GrpE has been shown to substantially increase quantities of recombinant proteins that are toxic or otherwise difficult to produce. The pOFXT7KJE3 plasmid has been used successfully for this purpose in ATP synthase subunit c expression .

What purification strategies yield the highest purity of recombinant ATP synthase subunit c?

Obtaining highly purified ATP synthase subunit c requires a strategic purification approach due to the protein's hydrophobic nature. Based on published methodologies, the following purification strategy has proven effective:

• Initial capture: For MBP fusion proteins, amylose affinity chromatography provides an excellent first purification step, taking advantage of the specific binding between MBP and immobilized amylose.

• Proteolytic cleavage: After initial purification, the fusion protein is treated with a specific protease (e.g., Factor Xa) to separate the c-subunit from its fusion partner.

• Reversed-phase chromatography: The cleaved c-subunit is then purified using reversed-phase column chromatography, which effectively separates the hydrophobic c-subunit from other proteins and the cleaved tag.

This sequential purification approach has yielded significant quantities of highly purified c₁ subunit with the correct alpha-helical secondary structure .

How can researchers verify the secondary structure of purified recombinant ATP synthase subunit c?

Verification of the correct secondary structure is crucial to ensure that the recombinant protein maintains its native conformation. Several complementary techniques can be employed:

• Circular Dichroism (CD) spectroscopy: This technique is particularly effective for assessing alpha-helical content, which is a critical structural feature of ATP synthase subunit c. CD spectra in the far-UV range (190-250 nm) provide quantitative measurements of secondary structure elements.

• Fourier Transform Infrared (FTIR) spectroscopy: FTIR can provide additional confirmation of secondary structure elements through analysis of amide bands.

• Limited proteolysis: Comparing the proteolytic digestion pattern of recombinant and native proteins can indicate whether the recombinant protein maintains the correct folding and accessibility of cleavage sites.

Research has confirmed that properly purified recombinant c₁ subunit displays the correct alpha-helical secondary structure, validating the expression and purification methodology .

How can recombinant ATP synthase subunit c be used to study c-ring assembly and stoichiometry?

The recombinant expression of ATP synthase subunit c provides a powerful platform for investigating c-ring assembly and the factors that influence ring stoichiometry. Several experimental approaches can be employed:

• In vitro reconstitution: Purified recombinant c-subunits can be used in reconstitution experiments to form c-rings in artificial membrane systems. This allows researchers to study the assembly process under controlled conditions and to analyze factors that affect ring formation.

• Site-directed mutagenesis: The recombinant expression system enables the introduction of specific mutations to investigate how particular amino acid residues influence c-ring assembly and stoichiometry. This approach can help identify key residues involved in subunit-subunit interactions.

• Cross-linking studies: Chemical cross-linking combined with mass spectrometry can be used to analyze the spatial arrangement of c-subunits within the ring and to determine ring stoichiometry.

These approaches address the fundamental question of why the number of c-subunits per oligomeric ring (c(n)) varies among organisms, which directly affects the ratio of protons translocated to ATP synthesized .

Why is c-ring stoichiometry an important area of research in ATP synthase studies?

The stoichiometry of the c-ring is a critical parameter in understanding the bioenergetics of ATP synthesis in different organisms. Research interest in this area stems from several key points:

• Bioenergetic efficiency: The number of c-subunits in the ring directly determines how many protons must be translocated to synthesize one ATP molecule. This ratio (H⁺/ATP) defines the thermodynamic efficiency of the enzyme and has significant implications for cellular energy metabolism.

• Evolutionary adaptation: Variations in c-ring stoichiometry among different species may reflect evolutionary adaptations to specific environmental conditions or metabolic requirements.

• Structure-function relationships: Understanding the molecular basis of c-ring stoichiometry variation provides insights into the fundamental principles governing protein assembly and the structure-function relationships in rotary enzymes.

The ratio of protons translocated to ATP synthesized varies according to the number of c-subunits per oligomeric ring, which is organism-dependent. Although this ratio is inherently related to the metabolism of the organism, the exact cause of the c(n) variability is not well understood, making it an important area for continued research .

What molecular biology techniques can be applied to recombinant ATP synthase subunit c for structure-function studies?

The development of recombinant expression systems for ATP synthase subunit c enables the application of powerful molecular biology techniques that cannot be applied to native c-rings. These include:

• Alanine scanning mutagenesis: Systematic replacement of residues with alanine to identify amino acids critical for function or assembly.

• Introduction of non-natural amino acids: Site-specific incorporation of non-natural amino acids with special properties (e.g., photo-crosslinkable, fluorescent) to probe specific aspects of structure and function.

• Fusion with reporter proteins or tags: Strategic fusion with fluorescent proteins or specific tags for tracking assembly, localization, or protein-protein interactions.

• CRISPR/Cas9 genome editing: For in vivo studies, CRISPR/Cas9 can be used to introduce specific mutations in the native gene to study effects on ATP synthase function in the organism.

These techniques provide researchers with unprecedented tools to investigate factors that influence the stoichiometric variation of the intact ring and to understand the molecular mechanisms of ATP synthase function .

How can researchers investigate the proton translocation mechanism in ATP synthase using recombinant subunit c?

Understanding the proton translocation mechanism is a fundamental research goal in ATP synthase studies. Recombinant subunit c provides several experimental approaches:

• Reconstitution in liposomes: Purified recombinant c-subunits can be reconstituted into liposomes along with other ATP synthase components to create a minimal functional system for studying proton translocation.

• Site-directed mutagenesis of proton-binding residues: Specific mutations can be introduced to alter residues involved in proton binding and release, allowing detailed investigation of the proton translocation pathway.

• Isotope labeling and NMR spectroscopy: Recombinant expression allows for isotopic labeling of specific residues, enabling high-resolution NMR studies of proton binding sites and conformational changes.

• Computational modeling: Structural data from recombinant proteins can inform molecular dynamics simulations to model the proton translocation process at the atomic level.

These approaches contribute to our understanding of how proton movement across the thylakoid membrane is coupled to ATP synthesis, a process that is fundamental to photosynthetic energy conversion .

What are common issues encountered when expressing ATP synthase subunit c and how can they be resolved?

Researchers working with recombinant ATP synthase subunit c commonly encounter several challenges that can be addressed with specific strategies:

The co-expression of chaperone proteins (DnaK, DnaJ, and GrpE) has been shown to substantially increase quantities of recombinant proteins that are toxic or otherwise difficult to produce. This approach has been successful with ATP synthase subunit c when using the pOFXT7KJE3 plasmid in conjunction with the expression vector .

How can researchers optimize the yield and quality of recombinant ATP synthase subunit c?

Optimizing recombinant protein production requires careful consideration of multiple factors throughout the expression and purification process:

• Expression optimization:

  • Fine-tune induction conditions (IPTG concentration, temperature, duration)

  • Test different E. coli strains (BL21, Rosetta, etc.)

  • Evaluate different fusion partners (MBP, GST, SUMO, etc.)

  • Implement codon optimization for the expression host

• Growth conditions:

  • Modify media composition (rich vs. minimal, supplementation)

  • Control growth rate (temperature, aeration)

  • Optimize cell density at induction

• Purification refinement:

  • Test different column matrices and conditions

  • Optimize buffer composition (pH, salt, additives)

  • Consider on-column refolding for proteins recovered from inclusion bodies

• Quality assessment:

  • Verify protein integrity by mass spectrometry

  • Confirm secondary structure by circular dichroism

  • Assess oligomeric state by size exclusion chromatography

By systematically addressing these factors, researchers have obtained significant quantities of highly purified c₁ subunit with the correct alpha-helical secondary structure, suitable for further structural and functional studies .