Recombinant Nocardioides sp. ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its fundamental role in cellular energetics?

ATP synthase subunit c, also known as subunit III in some nomenclature systems, is a critical component of the F₀ domain of ATP synthase. This highly conserved membrane protein forms a ring structure embedded in the membrane (thylakoid membrane in chloroplasts or inner mitochondrial membrane in eukaryotes) and plays a central role in the rotary mechanism of ATP synthesis. The c-subunit ring functions as a proton-translocating rotor that converts the energy of proton flow along an electrochemical gradient into mechanical rotation. This rotation is mechanically coupled to the γ-stalk in the F₁ region, driving the catalysis of ATP synthesis at the three catalytic sites in the F₁ domain .

The rotation of the c-subunit ring is directly coupled to proton translocation across the membrane. As protons move from the lumen to the stroma (in chloroplasts) or from the intermembrane space to the matrix (in mitochondria), they drive the rotation of the c-ring, which in turn rotates the γ-subunit. This mechanical rotation drives conformational changes in the β-subunits that catalyze the synthesis of ATP from ADP and inorganic phosphate .

How does the stoichiometry of ATP synthase c-subunits vary across different organisms?

The number of c-subunits forming the ring structure varies significantly across different organisms, ranging from 10 to 15 subunits per ring. This stoichiometric variation directly impacts the bioenergetic efficiency of ATP synthesis, as it determines the coupling ratio between proton translocation and ATP production. The coupling ratio (ions transported : ATP generated) ranges from 3.3 to 5.0 among organisms for which it has been determined .

This variation is significant because the number of ATP molecules generated per complete rotation of the c-ring is consistently 3 in all known ATP synthases, while the number of protons required to complete one rotation depends on the number of c-subunits in the ring . The purpose or evolutionary advantage of this stoichiometric variation remains undefined, despite various hypotheses being proposed .

What expression systems have been successfully used for recombinant production of ATP synthase subunit c?

Recombinant expression of ATP synthase subunit c has been successfully achieved using Escherichia coli expression systems. For example, the c₁ subunit from spinach (Spinacia oleracea) chloroplast ATP synthase has been produced in a recombinant E. coli expression system and subsequently purified in milligram quantities . This approach enables researchers to apply molecular biology techniques that cannot otherwise be used with native c-rings.

For effective expression, fusion protein approaches have proven valuable, particularly using the maltose binding protein (MBP) as a fusion partner. This strategy helps overcome the hydrophobic nature of the c-subunit, which otherwise tends to form inclusion bodies or exhibits toxicity to host cells. The recombinant approach with appropriate purification methods has been shown to yield significant quantities of highly purified c₁ subunit with the correct alpha-helical secondary structure .

What methodological approaches enable structural and functional studies of recombinant ATP synthase subunit c?

To conduct meaningful structural and functional studies of recombinant ATP synthase subunit c, researchers should employ a multi-faceted approach:

• Protein expression optimization: When expressing Nocardioides sp. ATP synthase subunit c, researchers should consider:

  • Codon optimization for E. coli expression

  • Use of fusion partners (MBP, thioredoxin, or SUMO) to enhance solubility

  • Temperature modulation during induction (typically lowered to 16-20°C)

  • Inducer concentration optimization (IPTG at 0.1-0.5 mM)

• Purification strategies:

  • Affinity chromatography utilizing the fusion tag

  • Size exclusion chromatography to separate monomeric from oligomeric forms

  • Ion exchange chromatography for final polishing

  • Detergent selection critical for maintaining native-like structure (common choices include n-dodecyl-β-D-maltoside or digitonin)

• Structural characterization:

  • Circular dichroism spectroscopy to confirm secondary structure integrity

  • NMR spectroscopy for detailed structural analysis

  • Cryo-EM for visualization of assembled c-rings

• Functional reconstitution:

  • Liposome reconstitution assays to test proton translocation activity

  • Patch-clamp studies to measure ion conductance properties

These methodological approaches must be carefully optimized for the specific properties of Nocardioides sp. ATP synthase subunit c, as membrane protein behavior can vary significantly between species .

How can researchers investigate the relationship between monomeric c₁ subunits and assembled c-rings?

Investigating the relationship between monomeric c₁ subunits and their assembly into functional c-rings requires specialized techniques that probe both structural transitions and oligomerization dynamics:

• In vitro assembly systems:

  • Controlled detergent-mediated reconstitution experiments

  • Lipid nanodisc assembly for maintaining native-like membrane environments

  • Time-course assembly studies with varying lipid compositions

• Analytical techniques:

  • Native-PAGE electrophoresis to distinguish between free c-subunit and assembled c-rings

  • Cross-linking experiments to capture assembly intermediates

  • Fluorescence resonance energy transfer (FRET) to monitor subunit interactions in real-time

• Mutagenesis approaches:

  • Strategic introduction of mutations at key interfaces

  • Creation of cysteine pairs for disulfide cross-linking

The reconstitution of multimeric c-rings from recombinant monomeric c₁ subunits is particularly valuable as it allows for the application of molecular biology techniques that cannot be applied to native c-rings. This capability is essential for studies investigating the factors that influence the stoichiometric variation of the intact ring .

What role does ATP synthase subunit c play in membrane permeability regulation and cell death pathways?

Beyond its canonical role in ATP synthesis, ATP synthase subunit c has been implicated in critical cellular processes related to membrane permeability and cell death:

• Mitochondrial Permeability Transition (MPT):

  • The c-subunit of FO ATP synthase is required for MPT, mitochondrial fragmentation, and cell death induced by calcium overload and oxidative stress

  • It appears to constitute a critical component of the Permeability Transition Pore Complex (PTPC)

  • Depletion of the c-subunit can prevent mitochondrial fragmentation in response to calcium ionophores like ionomycin

• Regulation of membrane leak conductance:

  • The c-subunit ring contains a regulated leak that modulates inner membrane ATP production efficiency

  • This leak may form or contribute significantly to the Cyclosporin A (CsA) regulated mitochondrial permeability transition pore (mPTP)

• Pathological implications:

  • Aberrant activity of the c-subunit-associated membrane permeability has been observed in certain pathological conditions

  • Free c-subunit levels (not incorporated into ATP synthase complexes) can be elevated in certain disease states, potentially contributing to mitochondrial dysfunction

These non-canonical functions highlight the multifaceted role of ATP synthase subunit c in cellular homeostasis and stress responses, suggesting potential therapeutic targets for conditions involving mitochondrial dysfunction .

How do the three genes encoding ATP synthase subunit c differ in expression and regulation?

ATP synthase subunit c is unique in being encoded by multiple genes that produce identical mature proteins but differ in their mitochondrial import sequences and regulatory mechanisms:

• Multiple encoding genes:

  • In humans and many mammals, three distinct nuclear genes (ATP5G1, ATP5G2, and ATP5G3) encode the c-subunit

  • All three genes produce polypeptides with different mitochondrial import sequences but form the identical mature protein after processing

• Differential expression patterns:

  • ATP5G2 has been identified as the dominant c-subunit gene in some tissues and cell types

  • Expression levels of the three genes can vary significantly across tissues and developmental stages

• Transcriptional and translational regulation:

  • Evidence suggests differential regulation of these genes at both transcriptional and translational levels

  • Some regulatory proteins may selectively bind to mRNAs of specific c-subunit genes; for example, FMRP (Fragile X Mental Retardation Protein) has been shown to bind ATP synthase β-subunit mRNA but not c-subunit mRNAs

• Pathological significance:

  • Altered ratios of gene expression have been observed in certain disease states

  • Elevated levels of ATP synthase components, including free c-subunit not incorporated into ATP synthase complexes, have been detected in some pathological conditions

Understanding the differential regulation of these genes may provide insights into tissue-specific energy requirements and adaptive responses to varying metabolic demands .

What are the optimal conditions for expressing recombinant Nocardioides sp. ATP synthase subunit c in E. coli?

Optimizing expression conditions for recombinant Nocardioides sp. ATP synthase subunit c requires careful consideration of several parameters:

Expression system optimization:

MBP fusion strategies have proven particularly effective for ATP synthase subunit c expression, as demonstrated with spinach chloroplast c₁ subunit. The fusion approach significantly improves solubility and reduces host toxicity while enabling purification via affinity chromatography .

Additionally, co-expression with molecular chaperones (GroEL/GroES) may further enhance proper folding and stability of the recombinant protein. For membrane integration studies, specialized E. coli strains with well-characterized membrane composition may be advantageous.

What purification strategies yield highest purity and structural integrity of ATP synthase subunit c?

Purification of recombinant ATP synthase subunit c presents unique challenges due to its hydrophobic nature and tendency to form aggregates. A multi-step purification strategy is recommended:

• Initial extraction:

  • Gentle cell lysis using sonication or French press

  • Membrane fraction isolation through differential centrifugation

  • Selective extraction using carefully optimized detergent mixtures (typically DDM, LDAO, or C₁₂E₈)

• Affinity chromatography:

  • For MBP fusion proteins: amylose resin affinity chromatography

  • Inclusion of appropriate detergent in all buffers (typically at 2-3× CMC)

  • Gradual detergent reduction during elution to promote proper folding

• Proteolytic cleavage and separation:

  • Site-specific protease treatment (Factor Xa, TEV, or SUMO protease)

  • Secondary affinity chromatography to remove cleaved fusion partner

  • Size exclusion chromatography to separate monomeric from oligomeric forms

• Final purification:

  • Ion exchange chromatography for removal of contaminants

  • Concentration using specialized membrane filters designed for hydrophobic proteins

• Quality assessment:

  • Circular dichroism to confirm alpha-helical secondary structure

  • Gel filtration analysis to confirm monodisperse preparation

  • Mass spectrometry to verify intact mass and sequence integrity

This multi-step approach has been successfully employed for c₁ subunit purification from spinach chloroplast ATP synthase and can be adapted for Nocardioides sp. ATP synthase subunit c .

How can researchers verify the proper folding and functionality of recombinant ATP synthase subunit c?

Verifying proper folding and functionality of recombinant ATP synthase subunit c requires multiple complementary approaches:

• Structural verification:

  • Circular dichroism (CD) spectroscopy to confirm the expected alpha-helical secondary structure

  • Thermal stability assays to assess protein stability

  • Limited proteolysis to probe the accessibility of cleavage sites

• Oligomerization assessment:

  • Native-PAGE analysis to detect formation of c-subunit oligomers

  • Cross-linking studies to trap and identify oligomeric states

  • Analytical ultracentrifugation to characterize assembly state

• Functional assays:

  • Reconstitution into liposomes for proton translocation measurements

  • Patch-clamp electrophysiology to measure ion conductance properties

  • Assembly assays with other ATP synthase components

• Interaction studies:

  • Binding assays with known interaction partners (e.g., a-subunit)

  • Co-immunoprecipitation of ATP synthase components

  • Surface plasmon resonance to quantify binding kinetics

For Nocardioides sp. ATP synthase subunit c, these methods should be optimized based on the specific properties of the protein, including detergent compatibility, buffer conditions, and expected oligomerization behavior .

How can recombinant ATP synthase subunit c be utilized for structural studies of complete c-rings?

Recombinant production of ATP synthase subunit c opens several avenues for structural studies of complete c-rings:

• In vitro reconstitution approaches:

  • Controlled assembly of c-rings in defined lipid environments

  • Systematic variation of lipid composition to study environmental effects on assembly

  • Introduction of specific mutations to probe assembly determinants

• Advanced structural biology methods:

  • Cryo-electron microscopy of reconstituted c-rings

  • Solid-state NMR studies of assembled rings

  • X-ray crystallography of stabilized c-ring complexes

• Hybrid approaches:

  • Combining recombinant subunits with native ATP synthase components

  • Cross-species chimeric c-rings to investigate stoichiometry determinants

  • Co-expression systems for coordinated assembly

The recombinant approach provides unique advantages for investigating the factors that determine c-ring stoichiometry, which varies across organisms and affects the bioenergetic efficiency of ATP synthesis. By studying how Nocardioides sp. ATP synthase subunit c assembles into rings compared to other species, researchers may gain insights into the evolutionary and functional significance of stoichiometric variations .

What insights can be gained from comparative studies of ATP synthase subunit c across different bacterial species?

Comparative studies of ATP synthase subunit c across different bacterial species offer valuable insights into evolution, adaptation, and functional diversity:

• Evolutionary analysis:

  • Sequence conservation patterns reveal functionally critical residues

  • Phylogenetic relationships between c-subunits correlate with metabolic adaptations

  • Identification of species-specific variations that may relate to environmental niches

• Structural comparisons:

  • Differences in c-ring stoichiometry (c₁₀ to c₁₅) across species

  • Species-specific structural adaptations for different membrane environments

  • Variations in proton-binding sites and ion selectivity

• Functional diversity:

  • Correlation between c-ring size and bioenergetic efficiency

  • Adaptations to different pH ranges and ion gradients

  • Integration with species-specific regulatory mechanisms

• Biotechnological applications:

  • Identification of c-subunits with enhanced stability for biotechnological applications

  • Engineering c-rings with altered stoichiometry for modified bioenergetic properties

  • Development of species-specific inhibitors for antibacterial applications

Studying Nocardioides sp. ATP synthase subunit c in comparison with other bacterial species may reveal unique adaptations related to the environmental niche of this soil bacterium, potentially offering insights into energy conservation strategies in diverse environments .

What are the challenges and solutions for reconstituting functional c-rings from recombinant monomers?

Reconstituting functional c-rings from recombinant monomeric c-subunits presents several significant challenges:

A key challenge is understanding the factors that determine the species-specific c-ring stoichiometry. The number of c-subunits per ring varies among organisms (from c₁₀ to c₁₅), affecting the bioenergetic efficiency of ATP synthesis . Successful reconstitution would allow researchers to investigate:

• The intrinsic properties of c-subunits that determine ring size

• The role of lipid environment in c-ring assembly

• The potential involvement of assembly factors in determining stoichiometry

• The dynamics of c-ring assembly and stability

These investigations are essential for understanding the purpose of stoichiometric differences across species and may reveal novel principles of membrane protein assembly .

How can researchers overcome expression toxicity issues with recombinant ATP synthase subunit c?

ATP synthase subunit c expression often faces toxicity issues in heterologous hosts due to its hydrophobic nature and tendency to integrate into membranes. Several strategies can overcome these challenges:

• Fusion protein approaches:

  • MBP fusion has proven particularly effective for ATP synthase subunit c expression

  • Other fusion partners worth exploring include thioredoxin, SUMO, and GST

  • C-terminal fusions may be preferable to N-terminal fusions depending on membrane topology

• Expression control strategies:

  • Tight regulation using repressible promoters (T7lac, araBAD)

  • Glucose suppression to prevent leaky expression

  • Reduced temperature cultivation (16-20°C)

  • Low inducer concentrations (0.01-0.1 mM IPTG)

• Specialized host strains:

  • C41(DE3) and C43(DE3) strains engineered for toxic protein expression

  • BL21(DE3) pLysS for stringent expression control

  • Lemo21(DE3) for tunable expression levels

• Membrane stress mitigation:

  • Co-expression of membrane integrase factors

  • Supplementation with specific phospholipids

  • Osmotic stabilizers in growth media

• Directed evolution approaches:

  • Selection for host adaptations that accommodate c-subunit expression

  • Screening for c-subunit variants with reduced toxicity

By implementing these strategies, researchers can significantly improve the expression yield and reduce toxicity when working with recombinant Nocardioides sp. ATP synthase subunit c .

What analytical techniques are most effective for detecting and quantifying ATP synthase subunit c oligomeric states?

Accurate detection and quantification of ATP synthase subunit c oligomeric states requires specialized analytical techniques:

• Electrophoretic methods:

  • Native-PAGE using digitonin or mild detergents (DDM, C₁₂E₉)

  • Clear-native PAGE for improved resolution of membrane protein complexes

  • Two-dimensional BN/SDS-PAGE for complex dissection

  • Ferguson plot analysis for molecular weight estimation

• Chromatographic approaches:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Native size exclusion chromatography using various detergent systems

  • Ion exchange chromatography under native conditions

• Biophysical techniques:

  • Analytical ultracentrifugation (sedimentation velocity and equilibrium)

  • Dynamic light scattering for hydrodynamic radius determination

  • Fluorescence correlation spectroscopy for diffusion coefficient measurement

• Microscopy methods:

  • Negative stain electron microscopy for direct visualization

  • Atomic force microscopy for topographical analysis

  • Single-molecule fluorescence for stoichiometry determination

• Chemical cross-linking coupled with mass spectrometry:

  • Identification of specific subunit-subunit interfaces

  • Determination of spatial relationships within the complex

  • Capture of transient assembly intermediates

Native-PAGE analysis has been successfully used to distinguish between free c-subunit and assembled ATP synthase, as demonstrated in studies examining ATP synthase assembly . This approach, combined with immunoblotting techniques, can provide quantitative information about the relative proportions of monomeric and oligomeric states.

How might genetic engineering of ATP synthase subunit c lead to novel biotechnological applications?

Genetic engineering of ATP synthase subunit c offers exciting possibilities for biotechnological applications:

• Bioenergetic engineering:

  • Modifying c-ring stoichiometry to alter the H⁺/ATP ratio

  • Creating chimeric c-rings with altered ion specificity (H⁺, Na⁺, K⁺)

  • Engineering thermostable variants for industrial applications

• Nanomotor development:

  • Harnessing the rotary motion of c-rings for nanoscale mechanical devices

  • Creating hybrid biological-synthetic nanomotors

  • Developing ATP synthase-based sensors and actuators

• Drug delivery systems:

  • Utilizing reconstituted c-rings as controllable membrane pores

  • Developing switchable nanopores for controlled release applications

  • Creating artificial vesicles with regulated permeability

• Bioremediation applications:

  • Engineering c-subunits with enhanced abilities to function in contaminated environments

  • Developing energy-generating bioremediation systems

  • Creating biosensors for environmental monitoring

• Bioelectronics integration:

  • Incorporating c-rings into bioelectronic interfaces

  • Developing ATP synthase-based bioelectronic circuits

  • Creating energy-harvesting bioelectronic devices

These potential applications build upon our understanding of the functional and structural properties of ATP synthase subunit c, including its role in proton translocation, its ability to form oligomeric rings, and its involvement in membrane permeability regulation .

What research questions remain unresolved regarding ATP synthase subunit c assembly and function?

Despite significant advances in understanding ATP synthase subunit c, several important questions remain unresolved:

• Stoichiometry determination factors:

  • What molecular mechanisms determine the species-specific number of c-subunits in a ring?

  • Are there assembly factors that influence c-ring stoichiometry?

  • How do membrane properties influence c-ring assembly and size?

• Assembly pathway elucidation:

  • What is the step-by-step process of c-ring assembly?

  • Do c-subunits assemble sequentially or through intermediate subcomplexes?

  • What is the role of lipids in facilitating proper assembly?

• Functional diversity exploration:

  • What is the evolutionary significance of varying c-ring sizes across species?

  • How do c-subunit variations contribute to adaptation to different environments?

  • Are there additional functions of the c-subunit beyond ATP synthesis?

• Regulatory mechanisms:

  • How is c-subunit expression regulated in response to energetic demands?

  • What post-translational modifications affect c-subunit function?

  • How do cells control the ratio of assembled versus free c-subunits?

• Pathological implications:

  • What is the precise role of c-subunits in mitochondrial permeability transition?

  • How does c-subunit dysfunction contribute to disease states?

  • Can c-subunit modulation be targeted therapeutically?

Addressing these questions will require integrated approaches combining structural biology, biochemistry, molecular biology, and advanced imaging techniques, potentially using Nocardioides sp. ATP synthase subunit c as a model system for comparative studies.