Recombinant Synechococcus sp. ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Synechococcus sp.?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the ATP synthase complex in Synechococcus sp. It forms a membrane-embedded ring structure comprising multiple c subunits. The c-subunit ring is essential for the mechanical coupling of proton translocation across the thylakoid membrane to ATP synthesis. In cyanobacteria, this ring enables the conversion of the proton gradient generated by photosynthesis or respiration into ATP production .

The gene encoding this protein in Synechococcus is known as atpE (with synonyms including atpH and SynRCC307_1882) . The c subunit has several name variants in the literature, including "ATP synthase F(0) sector subunit c," "F-type ATPase subunit c," and "Lipid-binding protein" .

How does the cyanobacterial ATP synthase differ from those in other organisms?

Cyanobacterial ATP synthases, such as those from Synechococcus sp., possess unique regulatory features not found in other organisms. The γ and ε subunits of the F0F1-ATP synthase display distinctive properties that regulate ATP hydrolysis activity, particularly to prevent wasteful ATP consumption during darkness .

A key difference is that the γ subunit of cyanobacterial F0F1 contains an extra amino acid segment (positions 198-222 in Synechocystis) whose deletion results in increased ATP hydrolysis activity. Additionally, the ε subunit strongly inhibits ATP hydrolysis activity through its C-terminal domain . These regulatory mechanisms help cyanobacteria maintain their intracellular ATP levels during prolonged dark periods, which is critical for their survival in natural environments with fluctuating light conditions .

Why is recombinant production of atpE important for cyanobacterial research?

Recombinant production of ATP synthase subunit c (atpE) enables:

• Structural studies: Obtaining sufficient quantities of purified protein for crystallographic or cryo-EM analysis

• Functional analyses: Investigating the mechanistic aspects of proton translocation and ATP synthesis

• Mutagenesis studies: Examining the effects of specific amino acid substitutions on function and regulation

• Reconstitution experiments: Rebuilding the multimeric c-ring to study its stoichiometry and assembly

• Development of biotechnological applications: Engineering cyanobacterial strains with enhanced bioenergetic capabilities

The recombinant approach allows researchers to apply molecular biology techniques that cannot be applied to native c-rings, providing new insights into the factors that influence the stoichiometric variation and functional properties of the intact ring .

What expression systems are most effective for producing recombinant Synechococcus sp. atpE?

Escherichia coli is the predominant expression system for recombinant ATP synthase subunit c from cyanobacteria due to its ease of genetic manipulation, low cost, and rapid growth. The most commonly used E. coli strain is BL21(DE3), which is deficient in certain proteases that might degrade the recombinant protein .

Several expression hosts can be considered:

• E. coli: Preferred for initial expression trials and high-throughput studies

• Yeast: Alternative for proteins requiring eukaryotic post-translational modifications

• Baculovirus: Used for proteins that are toxic to bacterial hosts

• Mammalian cells: Employed when complex folding or modifications are necessary

Expression vectors incorporating strong inducible promoters (like T7) and appropriate fusion tags (particularly 6×His for purification) have shown good results with cyanobacterial membrane proteins .

What are the optimal conditions for solubilization and purification of recombinant atpE?

Optimal conditions for handling recombinant ATP synthase subunit c include:

Solubilization:

• The hydrophobic nature of atpE requires careful solubilization

• Typically, a buffer containing glycerol (10-15%) and mild detergents helps maintain protein stability

• Common detergents include n-dodecyl β-D-maltoside (DDM) or digitonin

Purification:

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA columns is the method of choice for His-tagged proteins

• Purification under native conditions preserves the structural integrity

• Typical purity achieved is >90% as reported for commercial preparations

Storage:

• Store at -20°C for short-term or -80°C for long-term storage

• Working aliquots can be stored at 4°C for up to one week

• Repeated freezing and thawing should be avoided to prevent protein degradation

How can the yield and solubility of recombinant atpE be optimized?

Optimizing yield and solubility requires addressing several factors:

Expression conditions:

• Temperature: Lower temperatures (16-20°C) often improve folding of membrane proteins

• Induction: Using lower IPTG concentrations (0.1-0.5 mM) for longer induction periods

• Media composition: Enriched media like Terrific Broth can enhance biomass

• Codon optimization: Adapting the gene sequence to the expression host's codon bias

Fusion tags strategy:

• N-terminal fusion tags like MBP (maltose-binding protein) or SUMO can improve solubility

• C-terminal His-tags facilitate purification without interfering with N-terminal signal sequences

• Inclusion of appropriate linker sequences between the protein and tags

mRNA structure considerations:

• Optimizing the 5' UTR and N-terminal codons can significantly impact expression levels

• Avoiding strong mRNA secondary structures near the ribosome binding site

What methods are used to verify the functional integrity of recombinant atpE?

Several complementary approaches can verify functional integrity:

Biochemical assays:

• ATP hydrolysis activity measurements using colorimetric phosphate detection

• Reconstitution into liposomes to measure proton pumping

• ATP synthesis assays in proteoliposomes using acid-base transitions

Biophysical characterization:

• Circular dichroism (CD) spectroscopy to verify secondary structure

• Fluorescence-based assays to monitor conformational changes

• Native gel electrophoresis to assess oligomerization state

Functional complementation:

• Transformation of atpE-deficient strains to verify in vivo functionality

• Measurement of ATP synthesis/hydrolysis rates in thylakoid membranes

• Assessment of proton gradient formation using pH-sensitive dyes

Experimental data from Synechocystis sp. PCC 6803 (a related cyanobacterium) showed that thylakoid membranes from an ε subunit C-terminal deletion mutant exhibited ATP hydrolysis activities approximately 2-fold higher than wild-type, confirming the inhibitory role of the ε subunit's C-terminal domain .

How can recombinant atpE be used to study the stoichiometry of the c-ring?

Investigating c-ring stoichiometry requires specialized approaches:

Recombinant reconstitution:

• Purified recombinant c-subunits can be reconstituted in vitro to form c-rings

• The stoichiometry can be determined by mass measurements of intact rings

• Cross-linking experiments can stabilize the complex for analysis

Cryo-electron microscopy:

• Single-particle analysis of reconstituted c-rings

• Determination of the exact number of c-subunits per ring

• Correlation of stoichiometry with organisms' bioenergetic demands

Mass spectrometry approaches:

• Native mass spectrometry of intact c-rings

• Cross-linking mass spectrometry to identify subunit interfaces

• Hydrogen-deuterium exchange to probe structure and dynamics

The stoichiometry of the c-ring is particularly significant as it determines the H⁺/ATP ratio, which varies across species and affects the bioenergetic efficiency of the organism .

How can recombinant atpE be engineered to enhance cyanobacterial stress tolerance?

Engineering ATP synthase components can improve cyanobacterial stress tolerance:

Site-directed mutagenesis approaches:

• Targeting the C252 position in the ATP synthase α subunit (AtpA-C252F mutation) significantly improves high light and high temperature tolerance in Synechococcus elongatus PCC 7942

• This mutation can be combined with modifications to atpE to further optimize stress resistance

Experimental evidence:

• Introduction of the AtpA-C252F mutation into sucrose-producing strains significantly improved cell growth under high light conditions (300 μmol photons/m²/s) and elevated temperatures (45°C)

• A similar approach with an ethanologenic strain increased biomass production (OD₇₃₀ of 7.2 compared to 5.1 in the control) under high light conditions

Application in cell factories:

• Engineered ATP synthase components can be utilized to develop more robust cyanobacterial chassis for biotechnological applications

• These modifications allow cells to maintain photosynthetic efficiency under stress conditions, thereby improving production of target compounds

How does the regulatory mechanism of ATP synthase differ between light and dark conditions in Synechococcus?

ATP synthase regulation in Synechococcus involves sophisticated mechanisms that respond to light/dark transitions:

Light conditions:

• In light, photosynthesis generates a strong proton gradient across the thylakoid membrane

• This gradient drives ATP synthesis through the ATP synthase complex

• Under optimal light conditions, both γ and ε regulatory mechanisms are inactive, allowing maximal ATP production

Dark conditions:

• In darkness, the proton gradient weakens substantially

• The ε subunit's C-terminal domain adopts an inhibitory conformation that prevents wasteful ATP hydrolysis

• The γ subunit's inserted region (positions 198-222) facilitates entry into the ADP-inhibition state

• AtpΘ (encoded by atpT) accumulates during darkness to further prevent ATP hydrolysis

Experimental evidence:

• Thylakoid membranes from a C-terminally truncated ε mutant (εΔC) showed approximately 2-fold higher ATP hydrolysis activity than wild-type

• The same mutant exhibited reduced ATP synthesis rates (0.36 ± 0.16 μmol ATP·mg Chl⁻¹·min⁻¹) compared to wild-type (0.71 ± 0.29 μmol ATP·mg Chl⁻¹·min⁻¹) under high light (200 μmol photons·m⁻²·s⁻¹)

• After prolonged dark incubation, εΔC mutants showed lower intracellular ATP levels and reduced cell viability compared to wild-type strains

These findings demonstrate that the inhibition of ATPase activity is crucial for maintaining ATP levels during darkness, which is essential for cyanobacterial survival in natural environments with fluctuating light conditions .

What role does atpE play in the proton-to-ATP ratio and bioenergetic efficiency?

The c-subunit ring (composed of atpE proteins) is fundamental to determining the proton-to-ATP ratio:

Stoichiometric determinants:

• The number of c-subunits in the ring establishes how many protons must pass through the complex to generate one ATP molecule

• This stoichiometry varies between species and affects the bioenergetic efficiency

• In cyanobacteria, the stoichiometry must balance the demands of both photosynthetic and respiratory energy conversion

Thermodynamic considerations:

• A larger c-ring (more subunits) requires more protons per ATP but can operate with a smaller proton motive force

• A smaller c-ring requires fewer protons per ATP but needs a larger proton motive force

• This relationship represents an evolutionary adaptation to specific environmental niches

Research applications:

• Recombinant expression of atpE allows investigation of factors controlling c-ring assembly and stoichiometry

• Understanding these mechanisms could enable engineering of ATP synthases with altered H⁺/ATP ratios

• Such modifications could potentially improve the efficiency of bioenergetic processes in synthetic biology applications

How do 2-oxoglutarate and ATP/ADP ratio influence the PipX interaction network in relation to ATP synthase function?

The PipX interaction network in cyanobacteria responds to metabolic signals that are closely linked to ATP synthase function:

2-Oxoglutarate sensing:

• 2-Oxoglutarate (2-OG) is a key metabolic intermediate that signals nitrogen status

• High 2-OG levels disrupt PipX-PII complexes, allowing PipX to interact with NtcA

• This interaction activates genes involved in nitrogen assimilation, which requires ATP

ATP/ADP ratio effects:

• The ATP/ADP ratio directly regulates PipX-PII and PipX-NtcA complexes

• When ATP synthase is inhibited (by DCCD addition), a sharp drop in ATP levels occurs (to ~20% of previous levels within 5 minutes)

• This change in ATP/ADP ratio affects the PipX interaction network, with implications for nitrogen metabolism regulation

Experimental evidence:

• NanoBiT complementation assays in Synechococcus elongatus PCC 7942 demonstrated that PipX-PII complex formation is regulated by both 2-OG levels and the ATP/ADP ratio

• The addition of DCCD (an ATP synthase inhibitor) resulted in a 10-fold induction of luminescence in reporter strains, confirming the sensitivity of PipX-PII interactions to ATP levels

This interconnection highlights how ATP synthase activity and the resulting energy status coordinate with nitrogen metabolism through the PipX interaction network in cyanobacteria .

What are the challenges in reconstituting functional c-rings from recombinant atpE subunits?

Reconstituting functional c-rings faces several challenges:

Assembly obstacles:

• The highly hydrophobic nature of atpE complicates handling and assembly

• Obtaining the correct stoichiometry and orientation during assembly

• Ensuring proper interaction with other components of the F₀ domain

• Maintaining stability of the reconstituted complex in the absence of natural lipid environment

Methodological approaches:

• Detergent screening to identify optimal solubilization conditions

• Lipid nanodisc or liposome reconstitution to provide a membrane-like environment

• Careful pH and ionic strength control during reconstitution

• Stepwise assembly with other F₀ components to stabilize the complex

Functional verification challenges:

• Demonstrating proton conductance through the reconstituted c-ring

• Coupling the reconstituted c-ring with F₁ components to verify rotation

• Measuring ATP synthesis/hydrolysis activities in the reconstituted complex

Research on spinach chloroplast ATP synthase indicates that the development of a recombinant expression system for c-subunits enables molecular biology techniques that cannot otherwise be applied to native c-rings, allowing further studies on factors influencing stoichiometric variation of the intact ring .

How do the ε and γ subunits interact with atpE to regulate ATP synthase activity?

The regulatory mechanisms involving ε, γ, and the c-subunit ring are intricate:

ε subunit regulation:

• The C-terminal domain of the ε subunit adopts an inhibitory conformation in darkness

• This conformation physically blocks rotation of the c-ring, preventing ATP hydrolysis

• The ε subunit's N-terminal β sandwich domain interacts with the c-ring and is essential for proper F₀F₁ assembly

γ subunit regulation:

• The unique inserted region (positions 198-222) in cyanobacterial γ subunit enables frequent entry into an ADP-inhibition state

• This inhibition helps maintain cellular ATP levels in the dark

• The γ subunit acts as the central rotary shaft connecting the c-ring to the catalytic F₁ portion

Experimental evidence:

• In Synechocystis sp. PCC 6803, deletion of the γ inserted region (γΔ198–222) or truncation of the ε C-terminal domain (εΔC) results in higher ATP hydrolysis activity

• A double mutant with both modifications showed severely compromised ability to maintain ATP levels during prolonged darkness

• After 72 hours in darkness, the double mutant retained only ~15% of its initial intracellular ATP, compared to ~55% in wild-type

These findings demonstrate that both the ε and γ subunits are crucial for preventing wasteful ATP hydrolysis in the dark, with γ likely playing the more dominant role .

What is the role of AtpΘ (encoded by atpT) in regulating Synechococcus ATP synthase activity?

AtpΘ is a specialized regulator of cyanobacterial ATP synthase:

Expression patterns:

• AtpΘ is maximally expressed in darkness

• Expression is very low under optimal phototrophic growth conditions

• Glucose addition neutralizes dark-induced atpT transcript accumulation

• Addition of uncouplers or electron transport inhibitors restores high transcript levels in the dark even in the presence of glucose

Regulatory mechanisms:

• AtpΘ prevents ATP hydrolysis (reverse reaction) under unfavorable conditions

• Its regulation occurs primarily at the post-transcriptional level

• Two transcriptional regulators (cyAbrB1 and cyAbrB2) bind to the atpT promoter

• The histone-like protein HU binds to the 5'UTR of atpT

Physiological significance:

• AtpΘ helps maintain ATP levels during conditions that potentially weaken the proton gradient

• Its regulation appears connected to the status of the electron transport chain or proton gradient

• AtpΘ homologs are present in all available cyanobacterial genomes, indicating a conserved important function

This regulatory protein represents an additional layer of control over ATP synthase activity in cyanobacteria, complementing the regulation provided by the ε and γ subunits.

How do environmental factors influence the expression and assembly of ATP synthase components in Synechococcus?

Environmental factors significantly impact ATP synthase expression and assembly:

Light intensity effects:

• High light conditions increase the demand for ATP synthesis during photosynthesis

• ATP synthase components may be upregulated to meet increased energy demands

• Engineering the ATP synthase α subunit (AtpA-C252F) improves high light tolerance

• This modification enables growth at light intensities up to 300 μmol photons/m²/s

Temperature influences:

• Elevated temperatures affect membrane fluidity and protein folding

• The AtpA-C252F mutation confers tolerance to high temperatures (45°C)

• This suggests adaptability of ATP synthase assembly under temperature stress

Nutrient availability:

• Nitrogen limitation affects the PipX interaction network, which is sensitive to ATP/ADP ratios

• Phosphorus limitation has been linked to viral acquisition of host phosphate-binding proteins, potentially affecting ATP synthesis through altered phosphate homeostasis

• Carbon availability influences ATP synthase regulation, as shown by the effects of glucose on atpT expression

Combined stress response:

• The strategy of engineering ATP synthase components has been successfully applied to create more robust cyanobacterial cell factories

• A sucrose-producing strain with the AtpA-C252F mutation showed improved growth and productivity under combined high light and high temperature stress