Recombinant Arabidopsis thaliana ATP synthase subunit c, chloroplastic (atpH)

What expression systems are typically used for recombinant production of atpH?

Several expression systems have been successfully employed for recombinant production of atpH, with the most common being:

• E. coli expression systems: The most widely used approach due to simplicity and high yield. The atpH gene is typically cloned into vectors such as pET28a with an N-terminal His-tag for purification purposes .

• Pichia pastoris: This yeast expression system has been used for expression of other ATP synthase components and offers advantages for membrane proteins that may require eukaryotic post-translational modifications .

• Cell-free expression systems: For difficult-to-express membrane proteins like atpH, cell-free systems can sometimes overcome toxicity issues encountered in cellular systems.

For optimal expression in E. coli, researchers typically use BL21(DE3) strains with induction at lower temperatures (28°C instead of 37°C) to enhance proper folding of the membrane protein .

How can I purify recombinant atpH after expression?

Purification of recombinant atpH requires specialized techniques due to its hydrophobic nature and membrane association. A standard protocol includes:

• Cell lysis: Sonication (amplitude 35, 20 min with 30s on/off cycles) in the presence of protease inhibitors (PMSF 1mM and protease inhibitor cocktail) .

• Membrane fraction isolation: Centrifugation at 12,000g to remove cell debris, followed by ultracentrifugation at 200,000g for 1h at 4°C to separate the membrane fraction .

• Solubilization: The membrane pellet is solubilized using mild detergents such as n-dodecyl-β-D-maltoside (DDM) at 1-2% to extract membrane proteins.

• Affinity chromatography: For His-tagged protein, Ni-NTA affinity chromatography using imidazole gradient elution .

• Size exclusion chromatography: Final purification step to obtain homogeneous protein preparation .

Typical yields range from 3-6 mg of purified protein per liter of culture with purity >90% as determined by SDS-PAGE .

What is the role of atpH in ATP synthesis and how does it function in the chloroplast?

The atpH subunit is a critical component of the F₀ portion of the chloroplast ATP synthase (cpATPase), which couples ATP synthesis to the light-driven electrochemical proton gradient. Key functional aspects include:

• Proton translocation: The c-ring formed by multiple atpH subunits facilitates proton movement across the thylakoid membrane along the electrochemical gradient .

• Rotary mechanism: Proton movement through the c-ring causes it to rotate, which mechanically drives conformational changes in the F₁ catalytic portion, leading to ATP synthesis .

• Energy conversion efficiency: The number of c-subunits in the ring (stoichiometry) directly affects the ratio of protons translocated to ATP synthesized. In chloroplasts, the c-ring typically contains 14 subunits, as confirmed by high-resolution (2.3 Å) structural studies .

The cpATPase activity is optimal at physiological temperatures and significantly inhibited at cold temperatures (4°C), which can limit oxidative phosphorylation in plants exposed to cold conditions .

How does the recombinant atpH compare structurally and functionally to the native protein?

When properly expressed and purified, recombinant atpH retains the essential structural and functional characteristics of the native protein:

Structural comparison:

• Secondary structure: Both recombinant and native atpH maintain predominantly alpha-helical structures as confirmed by circular dichroism spectroscopy .

• Oligomerization: Recombinant c-subunits can assemble into rings similar to native ones, though the efficiency of assembly may depend on reconstitution conditions .

Functional aspects:

• The recombinant protein can be reconstituted into liposomes to measure proton translocation activity.

• When incorporated into membrane systems with other ATP synthase components, recombinant atpH can participate in ATP synthesis.

• Presence of purification tags may affect certain protein-protein interactions.

• Reconstitution of the complete functional c-ring from recombinant monomers remains challenging and may require specific chaperones like AtCGL160 .

What are the optimal conditions for reconstituting functional c-rings from recombinant atpH monomers?

Successful reconstitution of functional c-rings from recombinant atpH requires careful attention to several parameters:

Buffer composition:

• pH 7.5-8.0 buffer containing 50 mM Tris-HCl

• 100-150 mM NaCl to maintain physiological ionic strength

• 5-10% glycerol for stability

• 0.05-0.1% appropriate detergent (typically DDM)

Critical factors for successful reconstitution:

• Lipid composition: A mixture of DOPC, DOPE, and DOPG in a 6:3:1 ratio mimicking the chloroplast membrane environment improves reconstitution efficiency.

• Temperature cycling: Gradual temperature shifts between 4°C and 30°C can facilitate proper ring assembly.

• Co-factors: Addition of specific cardiolipins can enhance ring formation.

• CGL160 protein: Including the AtCGL160 protein significantly improves c-ring assembly efficiency, as this protein functions as a specific assembly factor for incorporating c-subunits into the cpATPase complex .

Successful reconstitution can be verified by:

• Negative-stain electron microscopy to visualize ring structures

• Blue native PAGE to assess oligomeric state

• Functional assays measuring proton translocation in proteoliposomes

How can I investigate the interaction between atpH and other ATP synthase subunits?

Several complementary techniques are effective for studying interactions between atpH and other ATP synthase components:

In vitro interaction studies:

• Co-immunoprecipitation (Co-IP): Using antibodies against atpH (such as Anti-AtpH ) to pull down interacting partners.

• Pull-down assays: Using His-tagged recombinant atpH to identify binding partners.

• Surface plasmon resonance (SPR): To determine binding kinetics and affinity constants.

In vivo interaction studies:

• Bimolecular fluorescence complementation (BiFC): To visualize protein interactions in plant cells.

• Förster resonance energy transfer (FRET): For detecting close proximity of fluorescently tagged proteins in living cells.

• Chemical cross-linking followed by mass spectrometry: To map interaction interfaces.

Recent research has revealed important interactions between atpH and AtCGL160, which is required for efficient incorporation of c-subunits into the cpATPase . The C-terminal portion of AtCGL160 is distantly related to prokaryotic ATP SYNTHASE PROTEIN1 (Atp1/UncI) proteins and is involved in c-ring assembly.

What approaches can be used to study the effects of mutations in atpH on ATP synthase function?

Systematic analysis of atpH mutations requires a multi-tiered approach:

Generation of mutations:

• Site-directed mutagenesis: To create specific amino acid substitutions in recombinant atpH.

• CRISPR/Cas9 genome editing: For introducing mutations in the native gene in Arabidopsis.

• Complementation of knockout lines: Expression of mutated versions in atpH-deficient plants.

Functional analysis methods:

• In vitro reconstitution: Comparing c-ring assembly efficiency between wild-type and mutant proteins.

• Proteoliposome assays: Measuring proton pumping activity and ATP synthesis rates.

• Chlorophyll fluorescence: Assessing effects on photosynthetic electron transport and energization of thylakoid membranes in vivo.

• Growth phenotyping: Evaluating effects on plant development under different environmental conditions.

A particularly informative approach is to focus on mutations in conserved residues that interact with other subunits or participate in proton translocation, such as those identified in the high-resolution (2.3 Å) structure of the spinach chloroplast c₁₄-ring .

How does temperature affect the activity of recombinant atpH and assembled ATP synthase?

Temperature has profound effects on ATP synthase function, with important implications for plant adaptation to environmental conditions:

Temperature effects on recombinant protein:

• Recombinant atpH shows optimal stability between 4-30°C during purification.

• Above 30°C, protein aggregation may occur, although some plant enzymes like PIMT2αω show maximal activity at 50°C despite 25°C being the normal growth temperature .

Temperature effects on assembled ATP synthase:
Research has revealed that cold temperature (4°C) specifically inhibits plant mitochondrial ATP synthase compared to other respiratory enzymes :

This temperature sensitivity:

• Cannot be overcome by cold-temperature acclimation of plants

• Provides a direct means of temperature perception by plant mitochondria

• May explain the evolution of alternative oxidase pathways that maintain respiratory rates at lower ATP synthesis efficiency during cold conditions

What role does atpH play in RNA editing in chloroplasts and how can this be studied?

Recent research has uncovered an unexpected role for ATP synthase components in RNA editing within chloroplasts:

ATPC1, the gamma subunit of ATP synthase in Arabidopsis chloroplasts, regulates editing at multiple sites of plastid RNAs, including the editing of atpH-3'UTR-13210 . This suggests a potential regulatory feedback between ATP synthase assembly and RNA processing.

Methods to study RNA editing associated with atpH:

• RNA immunoprecipitation (RIP): To identify RNAs associated with ATP synthase components.

• RNA editing site-specific assays: Using specific primers to amplify regions containing editing sites, followed by sequencing.

• Protein-protein interaction studies: To identify interactions between ATP synthase components and known RNA editing factors (MORFs, ORRM1, and OZ1).

The interaction between energy metabolism and RNA processing represents an emerging area of research. Disruption of ATPC1 increases editing at some sites (including atpH-3'UTR-13210) while decreasing editing at others, suggesting complex regulatory mechanisms .

How can I effectively use recombinant atpH for structural studies of the c-ring assembly?

Structural characterization of the c-ring requires specialized approaches due to its membrane-embedded nature:

Sample preparation methods:

• In meso crystallization: This technique has enabled high-resolution (2.3 Å) structure determination of the c₁₄-ring from spinach chloroplasts .

• Reconstitution into nanodiscs: Allows for a more native-like lipid environment for structural studies.

• Detergent screening: Identifying optimal detergents that maintain c-ring integrity.

Structural analysis techniques:

• X-ray crystallography: For high-resolution structural determination (requires crystallization).

• Cryo-electron microscopy: Increasingly used for membrane proteins without crystallization.

• Atomic force microscopy: For topological analysis of reconstituted c-rings in lipid bilayers.

• Solid-state NMR: For studying dynamics and interactions in membrane environment.

Key structural findings include the identification of circular electron densities inside the c-ring that may originate from isoprenoid quinones, suggesting these molecules might be universal cofactors of ATP synthases that stabilize the c-ring and prevent ion leakage .

What are the latest advances in understanding ATP compartmentation in relation to chloroplast ATP synthase function?

Recent research using FRET-based ATP sensors has revealed important insights into ATP compartmentation in Arabidopsis:

• Compartmental ATP concentrations: Plastidic ATP concentrations in cotyledon, hypocotyl, and root of 10-day-old seedlings are significantly lower than cytosolic concentrations .

• Developmental changes in ATP transport: Exogenous ATP can enter chloroplasts isolated from 4-5-day-old seedlings but not those from 10-20-day-old photosynthetic tissues, correlating with decreased expression of nucleotide transporters (NTTs) in mature tissues .

• Balancing ATP:NADPH ratios: Unlike diatoms, which import ATP into chloroplasts to support the Calvin-Benson-Bassham cycle, mature chloroplasts of Arabidopsis do not balance the ATP:NADPH ratio by importing ATP from the cytosol. Instead, they export surplus reducing equivalents to mitochondria, which then supply ATP to the cytosol .

These findings have important implications for understanding energy metabolism in plants and highlight the interdependence of chloroplasts and mitochondria in maintaining cellular energy homeostasis.

What are common challenges in expressing and purifying recombinant atpH and how can they be overcome?

Working with recombinant atpH presents several challenges due to its hydrophobic nature and membrane localization:

Common challenges and solutions:

• Low expression levels:

  • Use low-copy vectors such as pET28a with inducible promoters

  • Lower induction temperature to 28°C to improve protein folding

  • Include 0.1 mM ferrous sulfate in growth media to support assembly

  • Co-express with appropriate chaperones

• Protein insolubility:

  • Express as a fusion with solubility enhancing tags (MBP or SUMO)

  • Include 10 mM pyruvate in lysis buffer to stabilize the protein

  • Optimize detergent type and concentration (typically 1-2% DDM for extraction)

• Protein aggregation during purification:

  • Add 5-10% glycerol to all buffers

  • Maintain samples at 4°C throughout purification

  • Include 0.05-0.1% detergent in all buffers after solubilization

  • Consider purification under reducing conditions (add 1 mM DTT)

• Low purity:

  • Use two-step purification: affinity chromatography followed by size exclusion

  • Include additional washing steps with low imidazole concentrations

  • Consider ion exchange chromatography as an additional purification step

How can I verify the functionality of purified recombinant atpH?

Assessing the functionality of purified recombinant atpH requires multiple complementary approaches:

Structural verification:

• Circular dichroism (CD) spectroscopy: To confirm proper secondary structure (predominantly alpha-helical).

• Size exclusion chromatography: To assess oligomeric state and homogeneity.

• Native PAGE: To analyze c-ring assembly.

Functional assays:

• Reconstitution into proteoliposomes: Measuring proton translocation using pH-sensitive fluorescent dyes.

• Assembly with other ATP synthase components: In vitro reconstitution with F₁ components to assess ATP synthesis.

• Complementation studies: Testing whether the recombinant protein can rescue function in atpH-deficient systems.

Interaction studies:

• Binding assays with known interacting partners: Such as AtCGL160, which specifically facilitates c-subunit incorporation into the cpATPase complex .

• Co-immunoprecipitation with anti-AtpH antibodies: Available commercial antibodies show specific reaction with atpH from various species including Arabidopsis thaliana, Spinacia oleracea, and Nicotiana benthamina .

A successful functional verification would demonstrate that the recombinant protein can assemble into oligomers, interact with appropriate partners, and participate in proton translocation when reconstituted into membrane systems.

What are the best approaches to study the stoichiometry and assembly of the c-ring in Arabidopsis chloroplast ATP synthase?

Determining c-ring stoichiometry and assembly mechanisms requires specialized techniques:

Stoichiometry determination methods:

• Mass spectrometry of intact c-rings: To directly measure the mass of the complete ring.

• High-resolution structural studies: X-ray crystallography or cryo-EM to visualize the number of subunits (14 subunits have been confirmed in spinach chloroplast c-rings) .

• Cross-linking followed by SDS-PAGE: To analyze oligomeric states.

• AFM imaging of reconstituted rings: To count individual subunits.

Assembly pathway investigation:

• Pulse-chase experiments: To track the incorporation of newly synthesized subunits.

• Analysis of assembly intermediates: Using blue native PAGE combined with western blotting.

• Interaction studies with assembly factors: Particularly AtCGL160, which is specifically required for efficient incorporation of c-subunits into the cpATPase .

Research has revealed that the network of hydrogen bonds between subunits plays a crucial role in determining c-ring stoichiometry, with the high-resolution structure of the spinach chloroplast c-ring providing detailed insights into these intersubunit contacts .