Recombinant Nandina domestica ATP synthase subunit c, chloroplastic (atpH)

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

ATP synthase subunit c is an essential component of the F₀ complex of ATP synthase in chloroplasts. Structurally, the 81-amino acid protein folds into an α-helical hairpin that embeds in the thylakoid membrane. Multiple c-subunits assemble to form a ring (c₍ₙ₎) structure, which is critical for the rotational mechanism that drives ATP synthesis. The rotation of this c-subunit ring is mechanically coupled to ATP production and is powered by proton translocation across the thylakoid membrane along an electrochemical gradient .

The c-subunit's function is fundamentally linked to energy transduction - it converts the energy stored in the proton gradient into mechanical rotation that enables the catalytic synthesis of ATP in the F₁ portion of the complex. This represents a critical step in photosynthetic metabolism, as it produces the ATP required for carbon fixation and other cellular processes .

What expression systems are most effective for recombinant production of atpH protein?

E. coli expression systems have proven highly effective for recombinant production of atpH proteins, including Nandina domestica ATP synthase subunit c. The protein can be successfully expressed with an N-terminal His-tag for purification purposes . When designing expression constructs, codon optimization for E. coli is recommended, as demonstrated in similar studies with spinach chloroplast ATP synthase subunit c .

For optimal expression:

• Design a synthetic gene with E. coli-optimized codons

• Include appropriate terminal restriction sites for cloning

• Consider fusion partners (such as maltose-binding protein or His-tag) to improve solubility and facilitate purification

• Express in E. coli strains optimized for membrane protein production

Alternative expression systems such as the yeast Komagataella phaffii (formerly Pichia pastoris) may also be considered for secreted production, which could simplify purification procedures .

What are the most effective purification strategies for recombinant atpH?

Purification of recombinant atpH typically employs affinity chromatography utilizing fusion tags, most commonly His-tags that enable purification via immobilized metal affinity chromatography (IMAC) . For researchers seeking tag-free protein or minimal purification methods, alternative approaches are available:

Two-Stage Minimal Purification Process:

• Buffer exchange to adjust pH and salt concentration

• Q-membrane filtration to remove host cell proteins and DNA

This approach can yield 1-2 mg of protein with >60% purity from a 20 mL culture, sufficient for many analytical characterization assays .

For Higher Purity Requirements:

• Affinity chromatography using His-tag or MBP fusion

• Size exclusion chromatography

• Ion exchange chromatography as a polishing step

When working with membrane proteins like atpH, inclusion of appropriate detergents throughout the purification process is essential to maintain protein solubility and native structure .

What analytical techniques are most informative for confirming the correct folding of recombinant atpH?

Multiple complementary techniques should be employed to confirm proper folding of recombinant atpH:

• Circular Dichroism (CD) Spectroscopy: Essential for confirming the expected α-helical secondary structure. The c-subunit should exhibit characteristic minima at 208 and 222 nm, indicative of α-helical content .

• Size Exclusion Chromatography (SEC): Useful for assessing oligomeric state and homogeneity.

• Differential Scanning Calorimetry (DSC): Provides thermal stability information and can detect misfolded populations .

• Mass Spectrometry: For confirming molecular mass and sequence integrity.

• Fluorescence Spectroscopy: Can be used to monitor structural changes, particularly when coupled with environmental-sensitive probes .

Importantly, structural characterization should be performed in conditions that mimic the native membrane environment, typically using detergent micelles or lipid nanodiscs, as the protein's native structure is dependent on the hydrophobic environment .

How can researchers assess the propensity of atpH to form alternative conformations or aggregates?

The c-subunit of ATP synthase has been shown to exhibit amyloidogenic properties under certain conditions, with a propensity to shift from its native α-helical conformation to β-sheet structures that can lead to fibril formation . To assess this behavior in recombinant atpH:

• Thioflavin T (ThT) Fluorescence Assays: Monitor β-sheet formation kinetics through increased ThT fluorescence.

• Atomic Force Microscopy (AFM): Directly visualize potential fibril or oligomer formation.

• Fourier Transform Infrared Spectroscopy (FTIR): Detect secondary structure changes from α-helical to β-sheet conformations.

• Dynamic Light Scattering (DLS): Track aggregate formation and size distribution.

• Calcium-Dependent Structural Changes: Since c-subunit aggregation can be calcium-dependent, assess structural changes across various calcium concentrations using the techniques above .

Researchers should be particularly attentive to storage conditions, as temperature, pH, and ionic strength can significantly influence conformational stability. Protein solutions should be monitored for signs of aggregation through regular DLS measurements during storage .

How can the functional activity of isolated recombinant atpH be assessed?

While isolated atpH subunits don't catalyze ATP synthesis independently, several approaches can assess their functional integrity:

• Reconstitution into Liposomes/Proteoliposomes:

  • Incorporate purified atpH into lipid bilayers

  • Measure proton conductance across membranes

  • Assess ion channel activity using planar lipid bilayer electrophysiology

• Binding Assays with Partner Proteins:

  • Measure interaction with other ATP synthase components using surface plasmon resonance (SPR)

  • Assess complex formation via pull-down assays

• Structural Integration Assessment:

  • Evaluate the ability of recombinant atpH to assemble into c-rings

  • Use negative stain electron microscopy to visualize assembled complexes

• Calcium Binding Studies:

  • Investigate calcium-dependent structural changes using isothermal titration calorimetry

  • Monitor conformational shifts in response to calcium using fluorescence spectroscopy

Notably, the c-subunit has been shown to form ion-conducting pores in planar lipid bilayers when oligomerized, which can be measured using electrophysiological techniques .

What approaches can be used to study c-ring assembly from recombinant atpH monomers?

Studying c-ring assembly from recombinant monomers is challenging but can be approached through:

• In vitro Reconstitution:

  • Incorporate purified atpH into detergent micelles or nanodiscs

  • Gradually remove detergent to promote self-assembly

  • Vary lipid composition to identify optimal assembly conditions

• Analytical Ultracentrifugation:

  • Monitor formation of oligomeric species in real-time

  • Determine sedimentation coefficients for different assembly states

• Cross-linking Studies:

  • Use chemical cross-linkers to stabilize intermediates

  • Analyze cross-linked products by SDS-PAGE and mass spectrometry

• Electron Microscopy:

  • Negative stain EM to visualize assembled ring structures

  • Cryo-EM for high-resolution structural analysis of successfully assembled rings

• Native Mass Spectrometry:

  • Analyze intact c-ring complexes to determine subunit stoichiometry

  • Compare to known c-ring stoichiometries (ranging from c₁₀ to c₁₅)

The stoichiometry of c-rings varies across species, with coupling ratios (ions transported:ATP generated) ranging from 3.3 to 5.0. Understanding the factors that influence Nandina domestica c-ring stoichiometry would provide valuable comparative data .

How might recombinant atpH be used to investigate the variable c-ring stoichiometry observed across species?

The variable stoichiometry of c-rings across species (c₁₀ to c₁₅) remains an intriguing biological question. Recombinant atpH provides a powerful tool to investigate the factors determining this variation:

• Site-Directed Mutagenesis Studies:

  • Introduce mutations at key interface residues

  • Assess effects on ring size and stability

  • Compare with sequence differences between species with different stoichiometries

• Heterologous Expression of c-subunits:

  • Express atpH from multiple species in the same system

  • Compare assembly properties and resulting ring sizes

  • Identify sequence determinants of stoichiometry

• Hybrid Ring Formation:

  • Co-express native and mutant/tagged versions

  • Analyze resulting mixed rings to understand assembly principles

  • Determine if stoichiometry is determined during or after assembly

• Reconstitution Under Varying Conditions:

  • Test effects of lipid composition, pH, and ionic strength on ring formation

  • Investigate whether environmental factors influence stoichiometry

This research could help resolve the longstanding question of why different organisms maintain different c-ring stoichiometries and the potential evolutionary or physiological advantages this variation confers .

What insights might the potential amyloidogenic properties of atpH provide for understanding mitochondrial membrane permeabilization?

Recent research suggests the c-subunit of ATP synthase can undergo conformational transitions from α-helical to β-sheet structures under certain conditions, potentially contributing to membrane permeabilization phenomena like the mitochondrial permeability transition:

• Structural Transition Analysis:

  • Compare conditions triggering conformational change in recombinant atpH

  • Investigate calcium-dependence of β-sheet formation

  • Examine potential parallels with other amyloidogenic proteins (Aβ, α-synuclein)

• Membrane Permeabilization Studies:

  • Measure ion conductance in lipid bilayers containing recombinant atpH

  • Characterize pore formation properties and conductance levels

  • Compare with canonical permeability transition pore conductances

• Potential Disease Relevance:

  • Investigate interactions with known disease-associated proteins

  • Examine possible roles in neurodegenerative conditions

  • Explore whether plant chloroplastic atpH shares these properties with mitochondrial equivalents

This research direction connects the basic biology of ATP synthase components with potential pathological mechanisms, offering insights into both fundamental membrane protein behavior and disease processes .

What are the most common challenges in working with recombinant atpH and how can they be addressed?

Researchers commonly encounter several challenges when working with recombinant atpH:

Additional considerations include:

• Storing purified protein with at least 6% trehalose to maintain stability

• Avoiding repeated freeze-thaw cycles

• Reconstituting lyophilized protein carefully to prevent aggregation

For recombinant expression, consider fusion constructs that improve solubility and expression levels while facilitating purification, such as N-terminal His-tags or maltose binding protein fusions .

How can researchers distinguish between properly folded atpH and misfolded conformations?

Distinguishing properly folded atpH from misfolded conformations requires a multi-technique approach:

• Circular Dichroism (CD) Spectroscopy:

  • Native atpH should show characteristic α-helical spectra

  • Misfolded forms often display increased β-sheet content

  • Monitor at multiple temperatures to assess thermal stability

• Size Exclusion Chromatography:

  • Native monomeric protein should elute at expected molecular weight

  • Aggregates elute in the void volume

  • Multiple peaks may indicate heterogeneous conformational states

• Functional Reconstitution:

  • Properly folded protein should successfully integrate into liposomes

  • Misfolded protein typically forms insoluble aggregates or fails to integrate properly

• Protease Resistance Assays:

  • Compare digestion patterns between suspected native and misfolded forms

  • Native protein typically shows characteristic protected fragments

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence can report on tertiary structure

  • Binding of environment-sensitive dyes can distinguish folded states

Researchers should note that the c-subunit has been shown to adopt β-sheet conformations under certain conditions, particularly in the presence of calcium. This may represent an alternative functional state rather than simple misfolding in some contexts .