Recombinant Synechocystis sp. ATP synthase subunit c (atpE)

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

ATP synthase subunit c (atpE) is a critical component of the F0 sector of F0F1-ATP synthase in Synechocystis sp. The protein consists of 81 amino acids with the sequence MDSTVAAASVIAAALAVGLGAIGPGIGQGNASGQAVSGIARQPEAEGKIRGTLLLTLAFMESLTIYGLVIALVLLFANPFA . It functions as part of the membrane-embedded proton channel that facilitates proton translocation across the membrane during ATP synthesis and hydrolysis. In photosynthetic organisms like Synechocystis, the F0F1-ATP synthase complex plays a crucial role in energy conservation by utilizing the proton gradient generated during photosynthesis to synthesize ATP.

How does Synechocystis sp. atpE differ from ATP synthase subunit c in other organisms?

The ATP synthase subunit c in Synechocystis sp. shares structural similarities with other bacterial ATP synthase c subunits but has unique features associated with photosynthetic organisms. While the core function remains conserved, photosynthetic organisms like Synechocystis have developed specialized regulatory mechanisms for their ATP synthase complexes to adapt to fluctuating light conditions. The atpE subunit works in conjunction with unique regulatory elements in the γ and ε subunits that are specifically adapted for photosynthetic organisms . These adaptations allow for efficient energy conservation during both light and dark conditions, distinguishing it from ATP synthase complexes in non-photosynthetic organisms.

What are the optimal storage conditions for recombinant Synechocystis sp. atpE protein?

Recombinant Synechocystis sp. atpE protein should be stored at -20°C to -80°C immediately upon receipt . For long-term storage, aliquoting is essential to avoid repeated freeze-thaw cycles which can significantly degrade the protein. Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing is not recommended . The protein is typically supplied in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0, which helps maintain stability during storage .

What is the recommended protocol for reconstituting lyophilized recombinant atpE protein?

For optimal reconstitution of lyophilized recombinant atpE protein:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom.

• Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL.

• Add glycerol to a final concentration of 5-50% (recommended default: 50%).

• Create working aliquots to avoid multiple freeze-thaw cycles.

• Store reconstituted aliquots at -20°C to -80°C for long-term storage .

This protocol minimizes protein degradation and maintains functional integrity for experimental applications.

How can researchers effectively measure ATP hydrolysis activity of systems containing recombinant atpE?

Researchers can measure ATP hydrolysis activity of systems containing recombinant atpE through several established methods:

• Isolated thylakoid membrane assay: Compare ATP hydrolysis activities of isolated thylakoid membranes containing the protein of interest in controlled conditions. This can be done by measuring inorganic phosphate release or through coupled enzyme assays.

• Kinetic measurements: Determine parameters such as Km values (wild-type Synechocystis F0F1 shows a Km value of approximately 140 μM for ATP hydrolysis) and Vmax to assess enzymatic activity .

• Comparative analysis: When studying modified versions of atpE or the entire ATP synthase complex, always include wild-type controls under identical conditions for meaningful comparison of hydrolysis rates.

• Light/dark conditions control: Since the activity of ATP synthase from photosynthetic organisms varies significantly between light and dark conditions, researchers should carefully control lighting conditions during experiments .

The experimental setup should include appropriate buffers, temperature control (typically 30°C for Synechocystis), and pH monitoring systems.

What methods can be used to investigate the interaction between atpE and other ATP synthase subunits?

Several methodological approaches can be employed to investigate interactions between atpE and other ATP synthase subunits:

• Co-immunoprecipitation: Using antibodies against atpE or other subunits to pull down protein complexes and identify interacting partners.

• Cross-linking studies: Chemical cross-linking followed by mass spectrometry to identify proximity and interaction sites between subunits.

• Mutagenesis studies: Creating specific mutations in atpE and other subunits to identify critical residues for subunit interactions and function, similar to studies performed with the ε subunit .

• Single-molecule analysis: Techniques such as those used to study the α3β3γ complex can be adapted to investigate rotational dynamics and interactions between subunits .

• Cryo-electron microscopy: For structural determination of the entire complex and specific subunit interactions.

• Reconstitution experiments: Using purified recombinant subunits to reconstitute functional complexes and assess the contribution of individual components.

How does the atpE subunit contribute to the regulation of ATP synthase activity in Synechocystis sp.?

The atpE subunit (subunit c) of ATP synthase in Synechocystis sp. forms part of the F0 sector and contributes to regulation primarily through its role in the proton translocation pathway. While atpE itself is not the primary regulatory subunit, it works in concert with other regulatory subunits, particularly the γ and ε subunits that display unique properties in photosynthetic organisms .

The physiological significance of atpE in regulation becomes apparent when examining the entire F0F1 complex:

• Proton channel formation: Multiple copies of atpE form the c-ring structure that constitutes the proton channel in the membrane.

• Integration with regulatory subunits: AtpE's function is closely tied to the regulatory actions of the ε subunit, which strongly inhibits ATP hydrolysis activity. This inhibition is particularly important for maintaining ATP levels during dark periods .

• Environmental adaptation: The regulatory system involving atpE and other subunits helps cyanobacteria adapt to changing light conditions, preventing wasteful ATP hydrolysis in darkness and maintaining energy homeostasis .

Research has shown that proper regulation of the ATP synthase complex is essential for cell viability under fluctuating environmental conditions, highlighting the integrated nature of atpE's role within the regulatory framework.

What are the key differences in ATP synthesis vs. hydrolysis regulation in systems containing Synechocystis atpE?

ATP synthesis and hydrolysis in systems containing Synechocystis atpE are differentially regulated, which is critical for energy conservation in photosynthetic organisms:

The contrasting regulation of synthesis versus hydrolysis reflects an evolutionary adaptation of photosynthetic organisms to:

• Maximize ATP production during illumination

• Prevent wasteful ATP consumption during darkness

• Maintain cellular ATP homeostasis across light-dark transitions

This bidirectional regulation is crucial for the survival of cyanobacteria in environments with fluctuating light conditions.

What experimental approaches can be used to study the role of atpE in proton translocation across the thylakoid membrane?

Several sophisticated experimental approaches can be employed to study the role of atpE in proton translocation:

• Fluorescent pH indicators: Utilize pH-sensitive fluorescent dyes like 9-aminoacridine (AY) to monitor the formation of pH gradients across thylakoid membranes in wild-type versus atpE-mutant strains. This approach has been successfully used to demonstrate reduced pH gradient formation in regulatory subunit mutants .

• Site-directed mutagenesis: Create specific mutations in the conserved residues of atpE thought to be involved in proton binding and translocation, followed by functional analysis.

• Reconstitution in liposomes: Incorporate purified recombinant atpE (along with other F0 components) into liposomes to create a minimal system for studying proton translocation in isolation.

• Patch-clamp electrophysiology: Apply patch-clamp techniques to study proton currents through the c-ring in various experimental conditions.

• Hydrogen/deuterium exchange mass spectrometry: Use H/D exchange to identify regions of atpE involved in proton binding and translocation.

• Real-time ATP synthesis monitoring: Couple proton gradient measurements with real-time ATP synthesis analysis to correlate proton translocation efficiency with ATP production.

• Cryo-EM structural analysis: Compare structures of wild-type and mutant c-rings to identify conformational changes associated with proton binding and release.

These approaches, used in combination, can provide comprehensive insights into the molecular mechanisms of proton translocation mediated by atpE in the Synechocystis ATP synthase complex.

How does the function of atpE contribute to Synechocystis survival under various environmental stresses?

The function of atpE as part of the ATP synthase complex is crucial for Synechocystis survival under various environmental stresses:

• Light-dark transitions: As a component of the ATP synthesis machinery, atpE contributes to the organism's ability to maintain energy homeostasis during light-dark transitions. The proper regulation of ATP synthase/hydrolase activity is particularly important during extended dark periods .

• Energy conservation: The ATP synthase complex containing atpE enables efficient energy conservation during photosynthesis, which is critical for survival in fluctuating light environments. Research with regulatory subunit mutants has shown that proper regulation prevents wasteful ATP hydrolysis, helping maintain cellular ATP levels during stress conditions .

• pH stress adaptation: As part of the proton translocation machinery, atpE contributes to the cell's ability to manage internal pH, which is particularly important under acid stress conditions.

• Temperature fluctuations: The structural stability of the ATP synthase complex, including atpE, affects its functionality at different temperatures, influencing the organism's ability to survive temperature fluctuations.

Experimental evidence with related ATP synthase mutants has demonstrated lower cell viability under prolonged dark incubation compared to wild-type strains, highlighting the importance of proper ATP synthase function (including atpE) for stress survival .

What is the relationship between atpE function and photosynthetic efficiency in Synechocystis?

The relationship between atpE function and photosynthetic efficiency in Synechocystis represents a critical aspect of cellular bioenergetics:

Research with ATP synthase regulatory subunit mutants has shown that disruption of proper regulation affects not only ATP synthesis rates but also the formation of transmembrane pH gradients, indicating the interconnected nature of photosynthetic electron transport and ATP synthesis machinery .

What are the current state-of-the-art methods for studying the structure-function relationship of atpE in situ?

Advanced methods for studying the structure-function relationship of atpE in situ include:

• Cryo-electron microscopy (cryo-EM): Allows visualization of the ATP synthase complex at near-atomic resolution without removing it from its native membrane environment. This technique has revolutionized our understanding of the c-ring structure and its interaction with other subunits.

• Single-particle analysis: Computational methods combined with cryo-EM to determine high-resolution structures of heterogeneous populations of ATP synthase complexes.

• In situ crosslinking mass spectrometry: Identifies interaction sites between atpE and other subunits within the native membrane environment.

• Super-resolution microscopy: Techniques like STORM and PALM can visualize the distribution and organization of ATP synthase complexes containing atpE in thylakoid membranes.

• Förster resonance energy transfer (FRET): Measures distances between fluorescently labeled subunits to determine conformational changes during catalysis.

• Nuclear magnetic resonance (NMR): Provides atomic-level information about the structure and dynamics of atpE within membrane environments.

• Time-resolved structural methods: Captures transient states during the catalytic cycle, providing insights into the dynamic aspects of atpE function.

• Single-molecule rotation assays: Similar to those used with α3β3γ subcomplexes, these can be adapted to study rotational dynamics in complexes containing atpE .

These cutting-edge approaches, often used in combination, are pushing the boundaries of our understanding of ATP synthase function at the molecular level.

How can researchers effectively use recombinant atpE to reconstitute functional ATP synthase complexes in vitro?

Researchers can effectively use recombinant atpE to reconstitute functional ATP synthase complexes in vitro through the following methodological approach:

• Protein expression optimization:

  • Express recombinant atpE with appropriate tags (such as His-tag) in E. coli expression systems

  • Optimize expression conditions to maximize yield while maintaining proper folding

  • Consider co-expression with chaperones if misfolding occurs

• Purification strategy:

  • Use affinity chromatography (e.g., Ni-NTA for His-tagged proteins) followed by size exclusion chromatography

  • Maintain appropriate detergent concentrations to preserve membrane protein stability

  • Achieve >90% purity as verified by SDS-PAGE

• Reconstitution protocol:

  • Prepare liposomes with lipid compositions mimicking the native thylakoid membrane

  • Gradually remove detergent using biobeads or dialysis to incorporate atpE into liposomes

  • Co-reconstitute with other purified ATP synthase subunits in the correct stoichiometric ratios

• Functional verification:

  • Assess proton translocation using pH-sensitive fluorescent dyes

  • Measure ATP synthesis/hydrolysis activities using established enzymatic assays

  • Compare kinetic parameters with those of native complexes

• Structural confirmation:

  • Use negative-stain electron microscopy to verify proper assembly

  • Apply more advanced structural techniques (cryo-EM, AFM) to confirm native-like architecture

This approach allows researchers to study the specific contribution of atpE to complex assembly and function, as well as the effects of mutations or modifications on ATP synthase activity.

What are the current knowledge gaps regarding atpE function in Synechocystis, and what research questions remain unanswered?

Despite significant advances in understanding ATP synthase function, several knowledge gaps regarding atpE in Synechocystis remain:

• Molecular mechanism of c-ring rotation: The precise mechanism by which proton binding and release drives c-ring rotation in Synechocystis atpE remains incompletely understood.

• Species-specific adaptations: How the structure and function of atpE in Synechocystis differs from that in other cyanobacteria and photosynthetic organisms in relation to their specific ecological niches.

• Post-translational modifications: Whether atpE undergoes post-translational modifications that affect its function under different environmental conditions.

• Interaction specificity: The molecular determinants that ensure proper interaction between atpE and other subunits during ATP synthase assembly.

• Regulatory networks: How the expression and assembly of atpE is regulated in response to changing environmental conditions.

• Evolutionary origins: The evolutionary pathway that led to the specific adaptations of atpE in photosynthetic organisms.

Key unanswered research questions include:

• How does the stoichiometry of atpE in the c-ring affect the efficiency of energy conversion?

• What is the specific role of atpE in the assembly pathway of the complete ATP synthase complex?

• How do mutations in atpE affect the balance between ATP synthesis and hydrolysis under fluctuating light conditions?

• Can targeted modifications of atpE enhance photosynthetic efficiency for biotechnological applications?

What promising new technologies might advance our understanding of atpE function in the future?

Several emerging technologies hold promise for advancing our understanding of atpE function:

• Cryo-electron tomography: Will allow visualization of ATP synthase complexes in their native cellular context, revealing organizational principles and interactions with other cellular components.

• AlphaFold and other AI-based structure prediction: Will facilitate accurate prediction of protein-protein interactions and conformational changes during catalysis, guiding targeted experimental approaches.

• Single-molecule FRET with improved temporal resolution: Will capture short-lived conformational states during proton translocation and c-ring rotation.

• Optogenetic control of ATP synthase: Will enable precise temporal control of ATP synthase activity to study its physiological consequences.

• Expanded genetic code and non-canonical amino acids: Will allow incorporation of novel functional groups for mechanistic studies and crosslinking experiments.

• Mass photometry: Will provide insights into the stoichiometry and assembly pathways of ATP synthase complexes.

• Microfluidic devices coupled with real-time imaging: Will enable studies of ATP synthase function under rapidly changing environmental conditions.

• CRISPR-Cas9 genome editing with enhanced precision: Will facilitate more sophisticated genetic manipulations to study atpE function in vivo.

• In-cell NMR: Will allow structural studies of atpE in its native cellular environment.

These technological advances, combined with traditional biochemical and biophysical approaches, promise to significantly enhance our understanding of atpE function in the coming years.