ATP synthase subunit c (atpE) is a membrane-embedded component of the F₀ sector responsible for proton transport during ATP synthesis. In Nocardioides sp., the recombinant form (UniProt ID: A1SHI6) consists of 69 amino acids (1–69 aa) and includes an N-terminal His tag for purification . It plays a pivotal role in maintaining proton gradients essential for ATP production .
The recombinant subunit c facilitates structural analyses of F₀ sector assembly, particularly proton channel formation and rotor-stator interactions .
Mutagenesis studies using this protein have elucidated residues critical for proton translocation (e.g., Glu/Asp in transmembrane helices) .
Overexpression or knockdown of subunit c isoforms (P1, P2, P3) alters respiratory chain activity, as shown in HeLa cell models .
Aberrant c-subunit expression correlates with metabolic disorders, including Fragile X syndrome and neurodegenerative diseases .
KEGG: nca:Noca_1758
STRING: 196162.Noca_1758
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 .
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 .
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 .
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 .
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 .
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:
Pathological implications:
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 .
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:
Differential expression patterns:
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:
Understanding the differential regulation of these genes may provide insights into tissue-specific energy requirements and adaptive responses to varying metabolic demands .
Optimizing expression conditions for recombinant Nocardioides sp. ATP synthase subunit c requires careful consideration of several parameters:
Expression system optimization:
Parameter | Recommended Condition | Rationale |
---|---|---|
Host strain | C41(DE3) or C43(DE3) | Engineered for toxic/membrane protein expression |
Expression vector | pMAL-c5X or pET-based | MBP fusion (pMAL) enhances solubility |
Growth temperature | 18-22°C post-induction | Reduces inclusion body formation |
IPTG concentration | 0.1-0.2 mM | Lower concentrations reduce toxicity |
Growth media | TB supplemented with 1% glucose | Enhanced biomass and reduced leaky expression |
Induction OD₆₀₀ | 0.6-0.8 | Optimal cell density for induction |
Post-induction time | 16-20 hours | Extended time at lower temperature |
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.
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 .
Verifying proper folding and functionality of recombinant ATP synthase subunit c requires multiple complementary approaches:
Structural verification:
Oligomerization assessment:
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 .
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 .
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:
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 .
Reconstituting functional c-rings from recombinant monomeric c-subunits presents several significant challenges:
Challenge | Solution Approach |
---|---|
Maintaining native-like membrane environment | Use of carefully selected lipid compositions; nanodiscs or liposomes with defined properties |
Achieving correct stoichiometry | Controlled assembly conditions; templates or scaffolds to guide assembly |
Preventing non-native aggregation | Optimized detergent-to-protein ratios; step-wise detergent removal |
Confirming functional integrity | Proton conductance assays; rotation measurements using fluorescent probes |
Assembly with other ATP synthase components | Co-reconstitution strategies; step-wise assembly protocols |
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 .
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:
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 .
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.
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 .
Despite significant advances in understanding ATP synthase subunit c, several important questions remain unresolved:
Stoichiometry determination factors:
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:
Pathological implications:
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.