Recombinant Silicibacter pomeroyi ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) and what is its function in Silicibacter pomeroyi?

ATP synthase subunit c (atpE) in Silicibacter pomeroyi is a small hydrophobic membrane protein that forms part of the F0 sector of ATP synthase. It functions as a critical component in the proton-translocating machinery of the ATP synthase complex. Multiple copies of this protein assemble into a cylindrical c-ring structure that rotates as protons flow through the complex, driving the synthesis of ATP.

The protein is encoded by the atpE gene (locus tag SPO3235) and has several synonyms including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, and lipid-binding protein . As in other organisms, this subunit directly cooperates with subunit a in the proton pumping process essential for energy generation .

Expression System

Recombinant Silicibacter pomeroyi ATP synthase subunit c is typically expressed in E. coli expression systems . Commercial preparations commonly include an N-terminal His-tag to facilitate purification. The full-length protein (amino acids 1-74) can be successfully expressed in E. coli with high purity (>90% as determined by SDS-PAGE) .

Purification Protocol

While the search results don't detail the specific purification protocol for this protein, standard approaches for His-tagged membrane proteins would typically involve:

• Bacterial cell lysis (sonication or detergent-based methods)

• Membrane fraction isolation via differential centrifugation

• Membrane protein solubilization using appropriate detergents

• Affinity chromatography using Ni-NTA resin

• Optional additional purification via ion exchange or size exclusion chromatography

Storage and Handling

The purified protein is available in lyophilized powder form and requires special handling :

• Store at -20°C/-80°C upon receipt

• Aliquoting is necessary for multiple use to avoid freeze-thaw cycles

• For reconstitution, centrifuge the vial before opening and dissolve in deionized sterile water to 0.1-1.0 mg/mL

• Addition of glycerol (5-50% final concentration) is recommended for long-term storage

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

What expression systems are most effective for producing recombinant Silicibacter pomeroyi atpE?

Based on available data, E. coli represents the most established expression system for Silicibacter pomeroyi atpE . For optimal expression of this membrane protein, several factors should be considered:

Recommended Expression Parameters

The effectiveness of the E. coli expression system is evidenced by the successful commercial production of the recombinant protein with greater than 90% purity . For researchers seeking to express this challenging membrane protein, careful optimization of these parameters would likely yield the best results.

Bacterial vs. Eukaryotic Subunit c

In contrast to eukaryotic ATP synthase subunit c, the Silicibacter pomeroyi version is smaller (74 amino acids) and lacks mitochondrial targeting peptides that are characteristic of eukaryotic counterparts . In mammals, three isoforms of F1F0-ATP synthase subunit c exist, differing only in their mitochondrial targeting peptides while sharing identical mature peptides .

Functional Conservation

Despite structural differences between species, the fundamental function of subunit c in proton translocation is highly conserved. The Silicibacter pomeroyi subunit c likely participates in proton pumping through direct cooperation with subunit a, similar to what has been observed in other organisms .

Oligomeric Structure

In many well-characterized systems, subunit c assembles into a cylindrical c10 oligomer . While the exact stoichiometry in Silicibacter pomeroyi has not been definitively established, it likely forms a similar ring structure as part of the functional ATP synthase complex.

Evolutionary Significance

As a marine bacterium, Silicibacter pomeroyi's ATP synthase components may reflect adaptations to its specific environmental niche. Research on other marine bacteria suggests specialized adaptations in energy metabolism proteins to accommodate marine conditions .

What methodological approaches are most effective for studying ATP synthase subunit c function?

Multiple complementary approaches can be employed to investigate the function of ATP synthase subunit c from Silicibacter pomeroyi:

Biochemical and Biophysical Characterization

• Reconstitution into liposomes: Essential for measuring proton translocation activity

• Spectroscopic analysis: UV-Vis spectroscopy, circular dichroism, and fluorescence techniques to analyze structural properties

• Oligomerization studies: Size exclusion chromatography, native PAGE, or analytical ultracentrifugation to examine c-ring assembly

Structural Biology Approaches

• X-ray crystallography: Challenging but potentially revealing for high-resolution structural details

• Cryo-electron microscopy: Particularly valuable for membrane protein complexes like ATP synthase

• NMR studies: Potentially useful for examining dynamics of specific regions

Molecular and Genetic Techniques

• Site-directed mutagenesis: To probe the function of specific residues

• Complementation studies: Similar to those performed with mammalian ATP synthase subunit c isoforms, where expressing exogenous P1 or P2 rescued respective silencing phenotypes

• Cross-linking experiments: To investigate interactions with other ATP synthase subunits

Functional Assays

• ATP synthesis measurements: Using coupled enzyme assays similar to those described for other Silicibacter pomeroyi enzymes

• Proton pumping assays: Utilizing pH-sensitive dyes or electrodes

• Respiratory chain analysis: Important since subunit c knockdown has been shown to impair mitochondrial respiratory chain structure and function in other systems

Research on related proteins demonstrates the value of combined approaches. For example, studies on DddW (a DMSP lyase from Ruegeria pomeroyi) utilized biochemical, kinetic, and spectroscopic characterization to elucidate its mechanism .

How does ATP synthase subunit c contribute to energy metabolism in marine environments?

Silicibacter pomeroyi (also referred to as Ruegeria pomeroyi in some sources) is a model marine roseobacter, and its ATP synthase subunit c plays a crucial role in the organism's adaptation to marine environments:

Integration with Marine Bacterial Metabolism

ATP synthase functions as part of the larger bioenergetic system in Silicibacter pomeroyi, working in concert with the electron transport chain. The search results mention connections between ATP synthase and electron transport components like cytochrome c in marine bacteria . This integration is particularly important in marine environments where energy resources may be limited or fluctuating.

Relationship to Marine-Specific Metabolic Pathways

Silicibacter pomeroyi has evolved specialized metabolic pathways for marine environments, including the ability to metabolize dimethylsulfoniopropionate (DMSP), an abundant osmolyte produced by marine phytoplankton . While ATP synthase is not directly involved in DMSP metabolism, it provides the energy required for these specialized pathways through ATP production.

Adaptation to Environmental Conditions

Marine bacteria like Silicibacter pomeroyi can alter their metabolism in response to environmental conditions. For example, research has shown differential metabolite production by Ruegeria pomeroyi in response to DMSP . The ATP synthase complex, including subunit c, would be critical for maintaining energy homeostasis during these metabolic shifts.

Potential Role in Quorum Sensing and Cooperative Behavior

Studies have demonstrated that Ruegeria pomeroyi shows quorum sensing behavior and differential metabolite production in marine environments . The energy provided by ATP synthase would support these complex communal behaviors that may be advantageous in marine ecosystems.

What challenges exist in expressing and studying membrane proteins like ATP synthase subunit c?

Working with ATP synthase subunit c presents several technical challenges common to membrane protein research:

Expression Challenges

• Toxicity to host cells: Overexpression of membrane proteins can disrupt host cell membranes

• Protein folding issues: Ensuring proper folding in heterologous expression systems

• Low expression yields: Membrane proteins typically express at lower levels than soluble proteins

• Inclusion body formation: Requiring complex refolding procedures

Purification Obstacles

• Detergent selection: Finding appropriate detergents that maintain protein structure and function

• Protein stability: Maintaining stability once extracted from the membrane environment

• Aggregation tendencies: Particularly challenging for highly hydrophobic proteins like atpE

• Preserving native oligomeric states: Critical for functional studies of the c-ring

Analytical Difficulties

• Structural analysis limitations: Membrane proteins are notoriously difficult to crystallize

• Functional reconstitution: Recreating the proper membrane environment for activity assays

• Protein-protein interaction studies: Capturing native interactions with other ATP synthase subunits

Storage and Handling Considerations

Special storage requirements are necessary for atpE, including:

• Avoiding repeated freeze-thaw cycles

• Storing at -20°C/-80°C for extended storage

• Maintaining working aliquots at 4°C for limited periods (up to one week)

• Using appropriate buffer systems with stabilizing agents (glycerol is recommended)

Despite these challenges, successful expression and purification of recombinant Silicibacter pomeroyi atpE has been achieved , demonstrating that these obstacles can be overcome with appropriate techniques.

How can site-directed mutagenesis elucidate functional mechanisms of Silicibacter pomeroyi atpE?

Site-directed mutagenesis represents a powerful approach for investigating structure-function relationships in ATP synthase subunit c. Although the search results don't describe specific mutagenesis studies on Silicibacter pomeroyi atpE, a methodological framework can be established based on approaches used for related proteins:

Key Residues for Mutagenesis Analysis

Methodological Approach

• Primer design: Design primers containing desired mutations

• PCR-based mutagenesis: QuikChange or overlap extension PCR methods

• Verification: Sequence verification of mutant constructs

• Expression and purification: Using established protocols for the wild-type protein

• Functional characterization: Comparative analysis of wild-type and mutant proteins

Proven Value in Related Systems

The effectiveness of this approach is demonstrated in studies of other Ruegeria pomeroyi proteins. For example, research on the DMSP lyase DddW showed that substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121) abolished enzymatic activity, demonstrating their essential role in function .

Coupling with Spectroscopic Analysis

Mutations can be further analyzed using spectroscopic techniques. For instance, electronic absorption and electron paramagnetic resonance (EPR) studies revealed substrate interactions with the iron site in DddW . Similar approaches could elucidate mechanistic details of proton binding and translocation in atpE.

What is known about the oligomerization and assembly of ATP synthase subunit c in bacterial systems?

The assembly of ATP synthase subunit c into functional c-rings is a critical aspect of ATP synthase biogenesis. While the search results provide limited specific information about this process in Silicibacter pomeroyi, key insights can be drawn from related systems:

General Assembly Principles

In bacterial ATP synthase, multiple copies of subunit c assemble into a ring structure within the membrane. The search results mention that "subunit c is assembled in a cylindrical c10 oligomer" , suggesting that in many bacteria, including potentially Silicibacter pomeroyi, the c-ring consists of ten subunit c proteins.

Functional Significance of Proper Assembly

The importance of proper subunit c assembly is highlighted by studies in other systems showing that alterations in this process can severely impact ATP synthase function. The search results indicate that in mammals, "silencing any of the three subunit c isoforms individually resulted in an ATP synthesis defect" , demonstrating the non-redundant nature of these components for proper complex assembly.

Interactions with Other ATP Synthase Components

The c-ring interacts with other ATP synthase components, particularly subunit a, in the proton pumping process . This interaction is critical for the rotary mechanism of ATP synthesis, where proton translocation through the a/c interface drives rotation of the c-ring.

Impact on Respiratory Chain

Proper assembly of ATP synthase, including the c-ring, has broader implications for cellular energetics. Research has shown that "subunit c knockdown impaired the structure and function of the mitochondrial respiratory chain" , suggesting that defects in ATP synthase assembly can affect the entire bioenergetic system.

Research Gaps and Future Directions

To fully understand the oligomerization and assembly of ATP synthase subunit c in Silicibacter pomeroyi, further studies are needed, including:

• Cryo-EM structural analysis of the assembled ATP synthase complex

• Protein-protein interaction studies to identify assembly factors

• In vitro reconstitution experiments to study assembly kinetics

• Genetic studies to identify factors affecting c-ring formation in this marine bacterium