Recombinant Platanus occidentalis ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c (atpH) in chloroplasts forms a critical component of the F₀ sector of ATP synthase, embedded within the thylakoid membrane. This protein assembles into an oligomeric ring structure (c-ring) that serves as the rotor component of the enzyme complex. During photosynthesis, protons are pumped across the thylakoid membrane, generating an electrochemical gradient. As these protons flow back through the ATP synthase complex, they drive the rotation of the c-ring, which is mechanically coupled to the synthesis of ATP in the F₁ sector of the enzyme .

The c-subunit possesses a predominantly alpha-helical secondary structure, with hydrophobic regions that anchor it within the lipid bilayer. Each c-subunit contains a proton-binding site, typically involving a conserved carboxylic acid residue. The number of c-subunits in the ring varies between species, which directly affects the bioenergetic efficiency of ATP production by altering the proton-to-ATP ratio .

In Platanus occidentalis, the chloroplastic ATP synthase subunit c consists of 81 amino acids and maintains the highly conserved structure typical of this protein across plant species. The protein's sequence (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) reveals the characteristic hydrophobic regions essential for membrane integration and function .

How is recombinant Platanus occidentalis ATP synthase subunit c expressed and purified?

A successful approach involves expressing the protein in Escherichia coli using a fusion protein strategy. The recombinant Platanus occidentalis ATP synthase subunit c can be expressed with an N-terminal His-tag in E. coli expression systems . This approach facilitates subsequent purification steps and improves solubility of the highly hydrophobic membrane protein.

For other chloroplastic ATP synthase c-subunits, researchers have developed a system using maltose binding protein (MBP) as a fusion partner. In this method:

• The gene encoding the c-subunit is first codon-optimized for expression in E. coli

• The optimized gene is inserted into a plasmid vector downstream of an MBP coding sequence

• The fusion protein is expressed in BL21 derivative E. coli cells

• The soluble MBP-c fusion protein is purified using affinity chromatography

• The fusion protein is cleaved to separate the c-subunit from MBP

• The c-subunit is further purified using reversed-phase chromatography

This methodology yields significant quantities of highly purified protein with the correct secondary structure. The purified protein from Platanus occidentalis is typically supplied as a lyophilized powder that requires appropriate reconstitution before experimental use .

What are the key properties of recombinant Platanus occidentalis ATP synthase subunit c protein?

The recombinant Platanus occidentalis ATP synthase subunit c, chloroplastic (atpH) protein exhibits several key properties that are important for researchers to consider:

Physical and Chemical Properties:

• Amino acid length: 81 residues for the full-length protein

• Molecular weight: Approximately 8-9 kDa

• Secondary structure: Predominantly alpha-helical

• Hydrophobicity: Highly hydrophobic, with multiple membrane-spanning regions

• Purity: Commercial preparations typically exceed 90% purity as determined by SDS-PAGE

Storage and Handling Requirements:

• Storage temperature: -20°C to -80°C for long-term storage

• Buffer composition: Typically provided in Tris/PBS-based buffer with 6% trehalose at pH 8.0

• Reconstitution: Should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Stability: Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

• Avoid repeated freeze-thaw cycles

Functional Characteristics:

• Forms part of the proton-translocating component of ATP synthase

• Participates in the assembly of the c-ring structure

• Contains binding sites for protons that are essential for the rotational mechanism

• Interacts with other subunits of the ATP synthase complex

Understanding these properties is crucial for designing experiments that investigate the structure, function, and assembly of ATP synthase complexes in chloroplasts.

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

The expression of recombinant chloroplastic ATP synthase subunit c presents significant challenges due to its highly hydrophobic nature and potential toxicity to host cells. Several expression systems have been developed, each with specific advantages and limitations.

E. coli Expression Systems:

• BL21 derivative strains have been successfully used for heterologous expression

• Fusion protein approaches are particularly effective:

  • Maltose Binding Protein (MBP) fusion strategy has been demonstrated to improve solubility and reduce toxicity

  • His-tag fusion enables simplified purification via affinity chromatography

• Codon optimization of the gene sequence for E. coli expression is typically required

• Induction conditions must be carefully optimized to balance protein yield with host cell viability

Key Methodological Considerations:

• Vector selection: pMAL vectors have proven effective for MBP fusion approaches

• Induction parameters: Temperature, inducer concentration, and induction time significantly impact yield

• Cell lysis conditions: Gentle lysis methods help preserve protein structure

• Fusion protein cleavage: Specific proteases like Factor Xa or TEV protease can be used for tag removal

• Final purification: Reversed-phase chromatography effectively separates the hydrophobic c-subunit

While E. coli remains the most widely used system, other expression platforms have potential advantages:

• Cell-free translation systems can overcome toxicity issues

• Yeast expression systems may provide a more eukaryotic environment for folding

• Plant-based expression systems might offer more authentic post-translational modifications

The choice of expression system should be guided by the specific experimental requirements, including protein yield, purity needs, downstream applications, and available resources.

What analytical methods can be used to verify the structure and function of recombinant ATP synthase subunit c?

Verifying the structure and function of recombinant ATP synthase subunit c requires a combination of analytical approaches. These techniques help ensure that the recombinant protein maintains native-like properties essential for experimental validity.

Structural Analysis Techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Confirms the alpha-helical secondary structure characteristic of c-subunits

  • Can detect structural changes under different conditions (pH, temperature, detergent)

  • Provides quantitative estimation of secondary structure content

• Size Exclusion Chromatography (SEC):

  • Assesses oligomeric state and homogeneity

  • Can detect proper c-ring assembly when combined with multi-angle light scattering

• Mass Spectrometry:

  • Verifies protein mass and sequence integrity

  • Can detect post-translational modifications or proteolytic processing

  • Useful for hydrogen/deuterium exchange studies to probe structural dynamics

• Nuclear Magnetic Resonance (NMR):

  • Provides atomic-level structural information in solution

  • Can detect conformational changes upon protonation/deprotonation

  • Particularly useful for studying proton-binding sites

Functional Analysis Approaches:

• Reconstitution Assays:

  • Incorporation into liposomes or nanodiscs

  • Measurement of proton translocation using pH-sensitive fluorophores

  • Assessment of ATP synthesis when combined with F₁ components

• Binding Studies:

  • Interaction with other ATP synthase subunits

  • Proton-binding capacity measurement

  • Lipid interaction analysis

• Electron Microscopy:

  • Visualization of c-ring assembly

  • Assessment of incorporation into membranes

  • Structure determination at near-atomic resolution using cryo-EM

Researchers have confirmed that recombinant ATP synthase subunit c produced using the MBP fusion approach maintains the correct alpha-helical secondary structure as determined by CD spectroscopy, suggesting that this expression and purification strategy yields properly folded protein suitable for functional studies .

How does the stoichiometry of c-subunits in the c-ring affect ATP synthase efficiency in different organisms?

The stoichiometry of c-subunits in the ATP synthase c-ring is a critical determinant of bioenergetic efficiency and varies significantly between species. This variation directly affects the proton-to-ATP ratio, which is a fundamental parameter of cellular energy conversion.

The number of c-subunits (n) in the c-ring determines how many protons must be translocated to generate one molecule of ATP. This relationship can be expressed as:

$\text{H}^+/\text{ATP ratio} = n/3$

This is because the synthesis of one ATP molecule requires a 120° rotation of the central stalk, while each c-subunit contributes to a (360°/n) rotation when translocating one proton .

Species-Specific Variation:
Different organisms have evolved different c-ring stoichiometries:

• Bacterial ATP synthases: typically 10-14 c-subunits

• Mitochondrial ATP synthases: typically 8-10 c-subunits

• Chloroplast ATP synthases: typically 14 c-subunits

This variation reflects adaptation to different energetic environments and metabolic demands. Organisms with higher c-subunit numbers have higher H⁺/ATP ratios, making ATP synthesis possible under lower proton motive force conditions, albeit at reduced energetic efficiency .

Research Implications:
The exact causes of c-ring stoichiometry variation remain unclear and represent an important area of investigation. Several hypotheses include:

• Adaptation to different environmental energy availability

• Optimization for specific metabolic requirements

• Structural constraints on ring assembly

• Co-evolution with other components of energy metabolism

Investigating these questions requires the ability to manipulate c-subunit properties and assembly, for which recombinant expression systems like those developed for Platanus occidentalis ATP synthase subunit c are invaluable. By expressing modified c-subunits or combining c-subunits from different species, researchers can probe the factors governing c-ring assembly and stoichiometry .

What experimental approaches can be used to study the assembly of the c-ring in vitro?

Reconstitution Methods:

• Detergent-Mediated Reconstitution:

  • Purified c-subunits are solubilized in detergent micelles

  • Controlled detergent removal promotes self-assembly

  • Assembled rings can be visualized by electron microscopy or isolated by size-exclusion chromatography

  • Different detergents can significantly affect assembly efficiency and ring stability

• Lipid-Based Reconstitution Systems:

  • Incorporation into liposomes or nanodiscs

  • Provides a native-like membrane environment

  • Enables functional studies of proton translocation

  • Can be combined with other ATP synthase subunits to study complex assembly

• Cell-Free Expression with Artificial Membranes:

  • Direct expression into preformed lipid bilayers

  • Avoids potential misfolding during purification and reconstitution

  • Allows real-time monitoring of assembly

Analytical Techniques:

• Fluorescence Resonance Energy Transfer (FRET):

  • Labeled c-subunits can report on proximity and orientation

  • Enables real-time monitoring of assembly kinetics

  • Can detect intermediate assembly states

• Cross-linking Studies:

  • Chemical or photoreactive cross-linkers can capture assembly intermediates

  • Mass spectrometry analysis of cross-linked products reveals subunit interactions

  • Time-resolved cross-linking can map assembly pathways

• Native Mass Spectrometry:

  • Can detect oligomeric states directly

  • Provides information on complex stability and heterogeneity

  • Requires specialized sample preparation for membrane proteins

The availability of highly purified recombinant c-subunits, such as those from Platanus occidentalis, enables these experimental approaches by providing consistent starting material. By controlling the conditions of reconstitution, researchers can investigate the factors that influence c-ring assembly, including lipid composition, pH, ionic strength, and the presence of other protein subunits .

How can site-directed mutagenesis of ATP synthase subunit c be used to investigate proton translocation mechanisms?

Site-directed mutagenesis of ATP synthase subunit c provides a powerful approach to interrogate the molecular mechanisms of proton translocation and energy coupling in ATP synthase. By systematically altering specific amino acid residues, researchers can probe structure-function relationships at the atomic level.

Key Target Residues for Mutagenesis:

• Proton-Binding Site:

  • The conserved carboxylic acid residue (typically glutamate or aspartate) in the middle of the second transmembrane helix is critical for proton binding

  • Conservative mutations (E→D or D→E) can alter proton affinity without abolishing function

  • Non-conservative mutations (E→Q or D→N) typically eliminate proton binding and transport

• Surrounding Residues:

  • Polar residues that form the proton-binding pocket influence pKa and proton exchange kinetics

  • Hydrophobic residues that seal the proton pathway from the aqueous environment

  • Interface residues that interact with other ATP synthase components

• Helix-Helix Interaction Sites:

  • Residues involved in c-c subunit interactions affect ring stability and assembly

  • Residues at the interface with subunit a influence proton access channels

Experimental Approaches Using Mutants:

• Functional Assays:

  • ATP synthesis activity measurements with reconstituted complexes

  • Proton translocation assays using pH-sensitive fluorophores

  • Measurement of proton-motive force utilization efficiency

• Structural Investigations:

  • Effects of mutations on c-ring assembly and stability

  • Conformational changes detected by spectroscopic methods

  • Altered subunit interactions identified by cross-linking

• Biophysical Characterization:

  • Changes in proton binding affinity (pKa shifts)

  • Altered thermal stability of the c-ring

  • Modified lipid interactions

The recombinant expression system for Platanus occidentalis ATP synthase subunit c provides an excellent platform for generating and studying such mutants. The ability to produce significant quantities of purified protein facilitates comprehensive functional and structural analyses that can reveal the intricate details of how ATP synthase harnesses proton flow to drive ATP synthesis .

What role does ATP synthase subunit c play in plant responses to environmental stresses?

ATP synthase subunit c plays crucial roles in plant responses to environmental stresses, serving as both a target of stress effects and a component of adaptation mechanisms. Recent research has revealed that ATP synthase function is intimately linked to stress response pathways in plants.

Responses to Temperature Stress:

Low-temperature stress significantly impacts ATP synthase activity in plants. Under cold stress conditions:

• ATP synthase activity decreases, leading to reduced ATP content

• Upon return to normal temperature, ATP synthase activity recovers

• This dynamic regulation helps plants balance energy production with environmental conditions

• In rice, protein kinase CTB4a enhances cold tolerance by binding to the ATP synthase β subunit (atpB), thereby increasing ATP synthase activity and ATP content

Hormonal Regulation:

Plant hormones significantly modulate ATP synthase expression and activity:

• Ethylene (ETH) treatment upregulates mitochondrial ATP synthases, with peak expression typically occurring at 10 hours post-treatment

• Jasmonic acid (JA) generally increases expression of ATP synthase genes, with maximum induction at approximately 10 hours

• Salicylic acid (SA) also induces expression of many ATP synthase genes, with peak values around 6 hours

Notable induction levels include:

• HbMATPR3 was strongly induced by ethylene (122-fold increase) and salicylic acid (17-fold increase)

• HbMATP7-1 showed a 41-fold increase in expression after jasmonic acid treatment

Genetic Regulation and Adaptation:

ATP synthase genes contain numerous cis-regulatory elements related to stress response:

• Light-responsive elements are the most abundant, reflecting the critical role of ATP synthase in photosynthesis

• Hormone-responsive elements for ethylene, jasmonic acid, and salicylic acid signaling

• Stress-responsive elements for various abiotic and biotic stresses

These findings suggest that ATP synthase subunit c and other components of the ATP synthase complex are not merely passive targets of stress effects but are actively regulated as part of plant adaptation mechanisms. The ability to produce recombinant ATP synthase subunits, such as the Platanus occidentalis chloroplastic ATP synthase subunit c, provides valuable tools for investigating these regulatory mechanisms at the molecular level .