Recombinant Populus alba ATP synthase subunit c, chloroplastic (atpH)

What is the structural role of subunit c in chloroplastic ATP synthase of Populus alba?

The c subunit of ATP synthase in Populus alba chloroplasts forms a multimeric ring (c-ring) embedded in the thylakoid membrane. This c-ring plays a critical mechanical role in ATP synthesis by rotating when protons are translocated across the membrane along an electrochemical gradient. This rotation mechanically couples proton translocation to ATP synthesis. The c-ring consists of multiple c subunits arranged in a circular formation, with the number varying by organism and possibly affecting the proton-to-ATP ratio .

How does the c-ring stoichiometry affect ATP synthesis efficiency in chloroplasts?

The c-ring stoichiometry (number of c subunits per ring) directly affects the proton-to-ATP ratio during synthesis. This relationship can be quantified as:

$\text{H}^+/\text{ATP ratio} = \text{c subunits per ring} / 3$

The variability in c-ring stoichiometry is organism-dependent and inherently related to the metabolism of the organism. In different plant species, this stoichiometry may vary, influencing energetic efficiency. While the exact cause of this variability remains incompletely understood, it represents an adaptation to specific metabolic requirements .

What specific structural features distinguish Populus alba atpH from other plant species?

While spinach chloroplast ATP synthase c subunit has been well-characterized with confirmed alpha-helical secondary structure , comparative analysis of the Populus alba c subunit would require examination of:

Researchers should conduct circular dichroism spectroscopy on purified recombinant Populus alba c subunit to confirm proper alpha-helical secondary structure, similar to verification methods used for spinach c1 subunit .

What expression system optimizations are required for recombinant Populus alba atpH production?

For efficient recombinant production of Populus alba ATP synthase c subunit, researchers should implement:

• Codon optimization: Design a codon-optimized gene insert for the expression host (typically E. coli) to overcome codon bias issues.

• Fusion protein approach: Express the hydrophobic c subunit as a soluble fusion protein with maltose binding protein (MBP) to increase solubility and prevent aggregation.

• Vector selection: Use an expression vector with tight regulation capabilities, as demonstrated in successful spinach c1 subunit expression .

• Host strain selection: BL21 derivative E. coli strains are recommended for membrane protein expression.

• Induction conditions: Optimize temperature, IPTG concentration, and induction time to maximize protein yield while maintaining proper folding.

A methodical approach monitoring these parameters is necessary for achieving significant quantities of properly folded recombinant protein .

How can researchers overcome the hydrophobicity challenges when expressing membrane-embedded atpH?

The hydrophobic nature of the c subunit presents significant expression challenges that can be addressed through:

• MBP fusion strategy: Express the c subunit as an MBP-c1 fusion protein to increase solubility, as demonstrated in spinach c1 expression .

• Cleavage optimization: Develop a protocol for efficient cleavage of the fusion protein while preventing aggregation of the released c subunit.

• Detergent screening: Identify appropriate detergents that maintain the structural integrity of the c subunit following cleavage from MBP.

• Buffer composition: Optimize buffer conditions to maintain stability during and after cleavage.

• Temperature control: Conduct expression at reduced temperatures (16-20°C) to slow protein production and facilitate proper folding.

This systematic approach has proven successful in obtaining soluble expression of eukaryotic membrane proteins in E. coli systems .

What purification strategy is most effective for recombinant Populus alba atpH?

Based on successful strategies with other plant ATP synthase c subunits, a multi-step purification protocol is recommended:

• Initial capture: Affinity chromatography using the MBP tag to isolate the fusion protein.

• Fusion protein cleavage: Site-specific protease treatment to separate the c subunit from MBP.

• Reversed-phase chromatography: Purification of the cleaved c subunit using a reversed-phase column, which has proven effective for hydrophobic membrane proteins .

• Size exclusion chromatography: Optional final polishing step to ensure homogeneity.

This approach has yielded highly purified c1 subunit from spinach chloroplast ATP synthase and should be adaptable to Populus alba with species-specific optimizations .

How can researchers confirm the structural integrity of purified recombinant atpH?

Structural verification should include multiple complementary techniques:

• Circular dichroism spectroscopy: To confirm the expected alpha-helical secondary structure, as demonstrated with spinach c1 subunit .

• Mass spectrometry: For accurate molecular weight determination and identification of potential post-translational modifications.

• Limited proteolysis: To assess the folding status by examining protease accessibility patterns.

• Functional reconstitution: Assembly with other ATP synthase subunits to assess functionality.

• NMR analysis: For more detailed structural information if sufficient quantities of isotopically labeled protein can be produced.

Confirmation of correct alpha-helical structure is particularly critical as it indicates proper folding, which is essential for functional studies .

How can researchers investigate c-ring stoichiometry variation in Populus alba compared to other species?

To study c-ring stoichiometry variation, researchers should:

• Purify intact c-rings: Develop methods to isolate intact c-rings from Populus alba chloroplasts.

• Mass determination: Use mass spectrometry techniques to determine the precise number of c subunits per ring.

• Electron microscopy: Apply cryo-EM to visualize the c-ring structure and count subunits.

• Cross-linking studies: Use chemical cross-linking to stabilize the c-ring followed by analysis.

• Comparative genomics: Analyze sequence determinants across species that might influence stoichiometry.

This multi-method approach would help elucidate the undefined factors affecting c-ring stoichiometry and structure , possibly revealing Populus-specific adaptations.

What experimental approaches can determine proton translocation efficiency in recombinant Populus alba ATP synthase?

To measure proton translocation efficiency:

• Reconstitution in liposomes: Incorporate purified recombinant c subunits with other ATP synthase components in liposomes.

• pH gradient monitoring: Use pH-sensitive fluorescent dyes to monitor proton movement across the membrane.

• ATP synthesis assay: Measure ATP production rates under defined proton gradient conditions.

• Calculation of H⁺/ATP ratio: Determine the number of protons required per ATP synthesized.

• Comparison with native enzyme: Benchmark recombinant enzyme performance against native Populus alba ATP synthase.

These measurements would allow correlation between c-ring stoichiometry and energetic efficiency in the context of Populus alba's specific metabolic requirements.

How does ATP synthase subunit c vary across Populus species and what are the functional implications?

Researchers investigating interspecies variation should:

• Sequence alignment: Compare atpH sequences across multiple Populus species (P. alba, P. tremula, P. trichocarpa, etc.).

• Identification of variable regions: Map variable amino acids onto structural models to identify functionally significant differences.

• Expression of multiple variants: Produce recombinant c subunits from different Populus species using identical protocols.

• Functional comparison: Assess performance differences in ATP synthesis rate, proton translocation efficiency, and assembly properties.

• Correlation with ecological niches: Relate observed differences to the specific environmental adaptations of each species.

This comparative approach could reveal how evolutionary pressures have shaped ATP synthase function across the Populus genus, potentially correlating with metabolic adaptations to different habitats.

Can insights from Populus ATP uptake mechanisms inform ATP synthase research?

While ATP synthase produces ATP, understanding how Populus roots take up exogenous ATP provides valuable contextual information:

• Metabolic integration: Poplar roots can incorporate exogenous ATP at rates of approximately 83 ± 27 nmol ATP g⁻¹ fresh weight h⁻¹ .

• Phosphatase activity: The uptake process partially involves cleavage by phosphatases, as indicated by inhibition studies with molybdate .

• Position-specific labeling: Studies using position-specific labeled ATP (γ-³³P-ATP vs. α-³³P-ATP) reveal differential uptake patterns, suggesting complex processing mechanisms .

• Competitive inhibition: Inorganic phosphate (Pi) competes with γ-³³P-ATP uptake but not α-³³P-ATP uptake, further supporting the role of phosphatases in processing .

What approaches can overcome the challenges of studying membrane protein interactions in ATP synthase complexes?

Advanced techniques for studying membrane protein interactions include:

• Cross-linking mass spectrometry (XL-MS): Identifies interaction interfaces between subunits within the ATP synthase complex.

• Native mass spectrometry: Preserves non-covalent interactions for analysis of intact complexes.

• Single-particle cryo-electron microscopy: Provides structural information about the entire ATP synthase complex.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Maps dynamic regions and interaction surfaces.

• Molecular dynamics simulations: Models membrane-protein and protein-protein interactions within the complex.

These complementary approaches can provide a comprehensive understanding of how the c subunit interacts with other components within the ATP synthase complex.

How can functional c-rings be reconstituted from recombinant subunits?

Reconstitution of functional c-rings requires:

• Optimization of detergent conditions: Identify detergents that support c subunit self-assembly into rings.

• Lipid composition: Determine the optimal lipid environment that promotes proper assembly and function.

• Assembly monitoring: Use techniques like analytical ultracentrifugation or native PAGE to verify correct oligomeric state.

• Proton binding assessment: Confirm that reconstituted c-rings maintain proper proton-binding capabilities.

• Integration with other subunits: Develop methods to combine reconstituted c-rings with other ATP synthase components.

Successful reconstitution would provide a powerful platform for structure-function studies, including the investigation of factors affecting c-ring stoichiometry in Populus alba.

What are the most promising approaches for modulating ATP synthase efficiency in Populus species?

Future research directions might include:

These approaches would contribute to our fundamental understanding of ATP synthase function while potentially informing biotechnological applications in Populus species.

How can advanced imaging techniques enhance our understanding of ATP synthase dynamics in Populus chloroplasts?

Emerging imaging techniques offer new opportunities:

• Single-molecule FRET: To capture conformational changes during c-ring rotation.

• Super-resolution microscopy: For visualizing ATP synthase distribution in thylakoid membranes.

• High-speed atomic force microscopy: To observe c-ring rotation in real-time.

• In situ cryo-electron tomography: For visualizing ATP synthase in its native membrane environment.

• Correlative light and electron microscopy: To connect structural features with functional states.

These techniques could reveal dynamic aspects of ATP synthase function that are inaccessible through static structural studies, providing insights into the unique adaptations of Populus alba ATP synthase.

Data Table: Comparative Properties of ATP Synthase Subunit c Across Species

This table provides comparative reference points for researchers working with Populus alba ATP synthase subunit c, highlighting both established knowledge and areas requiring investigation.