Recombinant Solanum tuberosum ATP synthase subunit c, chloroplastic (atpH)

What expression systems are most effective for producing recombinant ATP synthase subunit c from Solanum tuberosum?

E. coli is the most widely used and effective expression system for recombinant ATP synthase subunit c from Solanum tuberosum. The highly hydrophobic nature of this membrane protein presents challenges that can be addressed through several strategies:

• Using a synthetic gene with codons optimized for E. coli expression significantly improves yield, as demonstrated in similar work with spinach chloroplast ATP synthase subunit c .

• Expression as a fusion protein, particularly with maltose binding protein (MBP) or His-tag, enhances solubility and facilitates purification .

• Alternative expression hosts include yeast, baculovirus, or mammalian cell systems, which may be considered for specific applications requiring eukaryotic post-translational modifications .

Expression parameters that significantly impact yield include:

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

A multi-step purification approach is necessary to achieve high purity recombinant ATP synthase subunit c:

• Initial extraction: Given its hydrophobic nature, effective solubilization requires appropriate detergents or chaotropic agents.

• Affinity chromatography: The primary purification step depends on the fusion tag used:

  • For His-tagged proteins, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

  • For MBP fusion proteins, amylose resin affinity chromatography

• Secondary purification: Size exclusion chromatography or ion exchange chromatography to remove contaminants and aggregates.

Using these approaches, researchers have achieved >90% purity as determined by SDS-PAGE . After purification, it's critical to verify that the protein maintains its native alpha-helical secondary structure using circular dichroism spectroscopy .

Storage in 50% glycerol at -20°C or -80°C stabilizes the protein, though repeated freeze-thaw cycles should be avoided. For working solutions, aliquots can be stored at 4°C for up to one week .

What techniques are most effective for studying the oligomerization of ATP synthase subunit c in vitro?

Understanding c-subunit oligomerization into functional c-rings requires multiple complementary approaches:

• Blue native PAGE: Preserves native protein interactions and can visualize intact c-rings.

• Chemical cross-linking: Coupled with mass spectrometry, identifies specific interaction interfaces between adjacent subunits.

• Analytical ultracentrifugation: Determines size, shape, and homogeneity of oligomeric assemblies.

• Electron microscopy: Cryo-EM provides high-resolution structural information of assembled c-rings.

• In meso crystallization: Particularly effective for membrane proteins, has been successfully used for c-rings from spinach chloroplasts .

• Atomic force microscopy: Visualizes c-rings in reconstituted membranes and can provide information on dynamics.

For successful oligomerization studies, the protein must be maintained in conditions that mimic the native membrane environment, typically using detergent micelles or lipid nanodiscs.

How does the amino acid sequence of Solanum tuberosum ATP synthase subunit c compare to other plant species?

The ATP synthase subunit c is highly conserved across plant species, reflecting its essential role. Comparison of the 81-amino acid sequences reveals:

The key differences are primarily in the N-terminal region, while the functionally critical regions involved in proton binding and translocation are almost perfectly conserved. These minor sequence variations may influence interactions with lipids or other subunits, potentially affecting c-ring assembly or stability.

How does the stoichiometry of the c-ring affect the bioenergetic efficiency of ATP synthesis?

The c-ring stoichiometry (number of c-subunits in the ring) directly determines the bioenergetic efficiency of ATP synthesis:

• Each c-subunit binds and transports one proton during rotation

• The complete 360° rotation of the c-ring drives synthesis of 3 ATP molecules at the catalytic sites in F1

• The H+/ATP ratio equals n/3, where n is the number of c-subunits

In chloroplasts, c-rings tend to have higher stoichiometries compared to other organisms:

How is ATP synthase activity regulated in response to light and dark conditions in chloroplasts?

Chloroplast ATP synthase activity is tightly regulated by light/dark transitions through multiple mechanisms:

• Redox regulation: A disulfide bond between two cysteine residues in the γ-subunit forms in the dark, inhibiting ATP hydrolysis activity to prevent wasteful ATP consumption. When exposed to light, this disulfide bond is reduced by thioredoxin, activating the ATP synthase .

• Proton gradient: Light-driven electron transport establishes the proton gradient that powers ATP synthesis. In the dark, this gradient dissipates, naturally limiting ATP synthase activity.

• Inhibitory proteins: Regulatory proteins similar to the mitochondrial inhibitor protein IF1 can bind to the F1 domain in the dark, preventing ATP hydrolysis. These dissociate under illumination when ATP synthesis is favored .

• Membrane energization: The ATP synthase requires specific energetic parameters for function, including a ΔpH close to 2 units and a p-side pH close to 6 .

These regulatory mechanisms ensure that ATP synthesis occurs primarily during illumination when photosynthetic electron transport is active, preventing futile ATP hydrolysis in the dark.

How does the expression and function of ATP synthase change under abiotic stress conditions?

ATP synthase plays a crucial role in plant responses to various abiotic stresses:

Drought stress: ATP synthase genes are often upregulated during drought, reflecting increased energy demand for stress response mechanisms. Overexpression of certain ATP synthase subunit genes improves drought tolerance .

Salt stress: In halophytic plants like Mesembryanthemum crystallinum, salt stress increases ATP content at NaCl concentrations up to 300 mM, suggesting enhanced ATP synthase activity. Similar enhancement of F-ATP synthase activity has been observed in wheat under high salt conditions .

Heat stress: Heat stress affects ATP synthase in complex ways:

• Some ATP synthase subunits show increased expression to potentially stabilize the complex

• Studies on subunit d show that its downregulation impairs ATP synthase function and increases heat sensitivity

• The ATP synthase complex may become unstable during recovery from heat stress

Cold stress: Low temperatures above freezing cause F-ATP synthase activity to decline more severely than other respiratory chain components. This leads to decreased ATP production and activation of alternative respiratory pathways .

The table below summarizes key findings related to ATP synthase subunits under stress conditions:

What role does ATP synthase subunit c play in mitochondrial permeability transition?

Recent research has identified ATP synthase subunit c as a potential channel component involved in mitochondrial permeability transition:

• The c-subunit ring has been proposed to form the pore of the mitochondrial permeability transition pore (mPTP) .

• Chloroform extraction of rat liver mitochondria yielded material with channel activity similar to mPTP, identified to contain ATP synthase c-subunit .

• The central pore of the c-ring could potentially serve as an ion channel when certain regulatory factors are present .

A "bent-pull" model has been proposed for c-subunit channel gating, where:

• Binding of specific factors to ATP synthase induces conformational changes in peripheral stalk subunits

• These changes modify interactions between the c-subunit and membrane embedded subunits

• Conformational changes pull a lipid "plug" or proteins from the c-subunit lumen, opening a channel

This model highlights both the importance of the c-subunit as a pore-forming component and the crucial role of other ATP synthase subunits (particularly e and g) in regulating pore formation .

How can recombinant ATP synthase subunit c be used to reconstitute functional c-rings in vitro?

Reconstitution of functional c-rings requires careful attention to experimental conditions:

• Solubilization: Purified recombinant subunit c must be solubilized in appropriate detergents (n-dodecyl-β-D-maltoside, n-octyl glucoside, or digitonin) that maintain native structure while allowing oligomerization.

• Assembly conditions: Optimal conditions include:

  • pH 6.5-8.0

  • Physiological ionic strength

  • Presence of specific lipids like phosphatidylcholine

  • Temperature control (typically 25-30°C)

• Verification methods:

  • Blue native PAGE to confirm oligomer formation

  • Electron microscopy to visualize ring structure

  • Circular dichroism to verify alpha-helical secondary structure

• Functional assessment:

  • Incorporation into liposomes

  • Measurement of proton translocation using pH-sensitive fluorescent dyes

  • Co-reconstitution with other ATP synthase subunits to form partial or complete complexes

This approach has been used successfully with spinach chloroplast ATP synthase c-subunit and provides a powerful tool for studying factors affecting c-ring stoichiometry and structure .

What are the challenges and solutions for studying interactions between ATP synthase subunit c and other components?

Studying ATP synthase subunit c interactions presents unique challenges due to its hydrophobic nature and membrane location:

Recent structural studies have revealed important interactions between the c-ring and other components:

• The c-ring interacts with central stalk subunits (γ, δ, and ε) to couple rotation to ATP synthesis

• In mitochondrial ATP synthase, the e subunit may be involved in c-ring channel gating

• The central cavity of the c-ring may contain specific lipids or proteins that affect its function

Understanding these interactions is crucial for elucidating the mechanism of ATP synthesis and the potential role of the c-subunit in other processes like mitochondrial permeability transition.