Recombinant Gloeobacter violaceus ATP synthase subunit c (atpE)

What is the amino acid sequence and structural characteristics of G. violaceus ATP synthase subunit c?

Gloeobacter violaceus ATP synthase subunit c (atpE) is an 82-amino acid protein with the sequence: MNDITAAASVIAAALAVGLAAIGPGIGQGNAASKAAEGIARQPEAEGKIRGTLLLSLAFMESLTIYGLLVSIVLLFANPFRG . This highly hydrophobic protein forms a hairpin-like structure with two membrane-spanning α-helices connected by a polar loop. The protein is characterized by conserved glycine motifs near the N-terminus that influence how closely c-subunits can pack together in the c-ring . The protein's hydrophobic nature necessitates special handling techniques during recombinant expression and purification.

Why is G. violaceus ATP synthase of particular interest to researchers?

G. violaceus holds special significance in evolutionary studies as it represents a primordial cyanobacterium that branched off from the main cyanobacterial evolutionary tree at an early stage . Unlike other cyanobacteria, G. violaceus lacks thylakoid membranes, with photosynthesis occurring directly in the cytoplasmic membrane . Its ATP synthase contains a c-ring with 15 subunits (c₁₅), which is on the higher end of the known c-ring stoichiometries . This unique evolutionary position makes G. violaceus ATP synthase valuable for studying the evolution of energy-converting systems and the functional significance of c-ring stoichiometry variations.

What expression systems are most effective for producing recombinant G. violaceus atpE protein?

The recombinant G. violaceus ATP synthase subunit c (atpE) can be effectively expressed in E. coli expression systems . Based on established protocols for similar ATP synthase c-subunits, the following approach is recommended:

• Clone the atpE gene into an expression vector with an N-terminal His-tag for purification

• Transform into an E. coli expression strain optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3))

• Grow cultures at 30-37°C until reaching OD₆₀₀ of 0.6-0.8

• Induce expression with IPTG (typically 0.5-1.0 mM) for 3-4 hours or overnight at a reduced temperature (16-20°C)

For difficult-to-express membrane proteins like atpE, fusion partners such as maltose-binding protein (MBP) can significantly enhance expression and solubility . Alternative expression systems such as cell-free systems may be considered for proteins that remain toxic to E. coli.

What purification strategy yields the highest purity and functional integrity of recombinant atpE?

A multi-step purification protocol is recommended for obtaining high-purity atpE protein:

• Cell lysis using sonication or pressure-based disruption in buffer containing protease inhibitors

• Membrane fraction isolation through differential centrifugation

• Solubilization of membrane proteins using appropriate detergents (e.g., DDM, LDAO)

• IMAC (Immobilized Metal Affinity Chromatography) using the His-tag

• Size exclusion chromatography as a polishing step

This approach typically yields protein with purity >90% as determined by SDS-PAGE . Critical parameters for maintaining functional integrity include:

How does the c₁₅ stoichiometry of G. violaceus ATP synthase affect its bioenergetic efficiency?

The c-ring stoichiometry directly determines the H⁺/ATP ratio of ATP synthase, which is a critical parameter for bioenergetic efficiency. G. violaceus possesses a c₁₅ ring, meaning that 15 protons must flow through the complex to generate 3 ATP molecules, resulting in an H⁺/ATP ratio of 5.0 .

This higher H⁺/ATP ratio has significant bioenergetic implications:

• More protons are required per ATP synthesized compared to organisms with smaller c-rings

• The enzyme can operate at lower proton motive force (pmf) values, potentially advantageous in energy-limited environments

• The thermodynamic efficiency may be lower compared to organisms with smaller c-rings (e.g., E. coli with c₁₀ or bovine mitochondria with c₈)

Mathematical modeling suggests that the thermodynamic efficiency (η) of ATP synthesis can be expressed as:

η = (ΔG_ATP)/(n·ΔG_H+)

Where n is the number of c-subunits, ΔG_ATP is the free energy of ATP synthesis, and ΔG_H+ is the free energy per proton . With all other parameters being equal, a higher n value (as in G. violaceus) results in lower thermodynamic efficiency but potentially greater robustness under low energy conditions.

What experimental approaches can be used to investigate c-ring assembly and stoichiometry?

Several complementary techniques can be employed to study c-ring assembly and stoichiometry:

• Atomic Force Microscopy (AFM): Provides direct visualization of c-ring diameter and subunit arrangement

• Cryo-Electron Microscopy: Enables high-resolution structural determination, as demonstrated with the 2.04 Å resolution structure of G. violaceus PSI

• Mass Spectrometry of Intact Complexes: Determines precise molecular weight of assembled c-rings

• Cross-linking coupled with SDS-PAGE: Reveals oligomeric states and subunit interactions

• Reconstitution experiments: Using recombinant c-subunits to form functional c-rings in vitro

For reconstitution experiments, purified recombinant c-subunits can be incorporated into liposomes along with other ATP synthase components to assess functional assembly. Successful approaches include:

• Detergent-mediated reconstitution followed by detergent removal

• Incorporation of purified c-subunits into lipid nanodiscs

• Co-expression of multiple ATP synthase components in a suitable host

These techniques can help determine how the primary sequence of the c-subunit influences ring formation and stoichiometry, which appears to be genetically encoded rather than environmentally regulated .

How can site-directed mutagenesis of G. violaceus atpE be used to investigate c-ring assembly determinants?

Site-directed mutagenesis offers a powerful approach to identify specific amino acid residues that determine c-ring stoichiometry and assembly. Based on comparative studies with other organisms, several key targets for mutagenesis in G. violaceus atpE include:

• N-terminal glycine motifs: Mutations in these regions can alter the packing of adjacent α-helices, potentially changing ring size

• Ion-binding glutamate residue: Essential for proton translocation and located in the second transmembrane helix

• Interface residues between adjacent c-subunits: Modifications can strengthen or weaken subunit-subunit interactions

A systematic mutagenesis approach should follow this workflow:

• Generate point mutations using PCR-based methods

• Express and purify mutant proteins

• Assess c-ring assembly in vitro

• Determine stoichiometry changes using techniques described in section 3.2

• Evaluate functional consequences through ATP synthesis assays

Mutagenesis studies have demonstrated that replacing the endogenous c-subunit gene with genes from other organisms results in c-ring stoichiometries matching the donor organism rather than the host , confirming that primary sequence is the key determinant of ring size.

What methods can be employed to study the interaction between G. violaceus atpE and other ATP synthase subunits?

Investigating subunit interactions is crucial for understanding ATP synthase assembly and function. Several approaches are particularly useful:

• Co-immunoprecipitation: Using antibodies against atpE or interaction partners to pull down protein complexes

• Yeast two-hybrid or bacterial two-hybrid assays: For detecting direct protein-protein interactions

• FRET (Förster Resonance Energy Transfer): For studying interactions in living cells

• Surface Plasmon Resonance (SPR): For quantitative measurement of binding kinetics

• GFP-tagging and fluorescence microscopy: For localization studies, similar to approaches used with FtsH proteases in cyanobacteria

When designing interaction experiments, researchers should consider:

These interactions are critical for understanding how the c-ring couples proton translocation to ATP synthesis through its rotational mechanism.

How does G. violaceus ATP synthase c-subunit compare to those from other photosynthetic organisms?

Comparative analysis reveals significant evolutionary insights about ATP synthase c-subunits across photosynthetic organisms:

The consistent finding of larger c-rings (c₁₃-c₁₅) in photosynthetic organisms compared to non-photosynthetic bacteria and mitochondria (c₈-c₁₀) suggests an evolutionary adaptation related to photosynthetic energy conversion . This adaptation may reflect the need to operate ATP synthase under the relatively lower and more variable pmf generated by photosynthetic light reactions compared to respiratory chains.

What insights can chimeric ATP synthases with G. violaceus components provide about c-ring assembly and function?

Chimeric ATP synthase studies offer powerful insights into structure-function relationships. By creating hybrid complexes containing components from different species, researchers can identify:

• Compatibility requirements between c-rings and other subunits

• Sequence determinants of c-ring stoichiometry

• Functional adaptations in different organisms

Experimental approaches include:

• Expressing G. violaceus atpE in heterologous hosts (e.g., E. coli, yeast)

• Creating domain-swapped chimeras between G. violaceus and other c-subunits

• Reconstituting ATP synthase with mixed subunit composition

Previous research has shown that replacing endogenous c-subunits with those from other organisms results in functional chimeric ATP synthases with c-ring stoichiometries matching the donor organism . This suggests that the primary sequence of the c-subunit is the predominant factor determining ring size.

For G. violaceus specifically, creating chimeras could help identify the molecular basis for its large c₁₅ ring. Strategic domain-swapping experiments between G. violaceus atpE and c-subunits from organisms with smaller rings (e.g., E. coli) could pinpoint the specific sequence regions responsible for the larger stoichiometry.

How might synthetic biology approaches utilizing G. violaceus atpE advance bioenergetic research?

Synthetic biology offers exciting possibilities for utilizing G. violaceus atpE in bioenergetic research:

• Designer c-rings with altered stoichiometry: Engineering c-subunits with modified interfaces could create ATP synthases with novel H⁺/ATP ratios, potentially optimizing energy conversion efficiency

• Biosensors: Creating fusion proteins between atpE and fluorescent reporters could enable real-time monitoring of proton flow or membrane potential

• Minimal ATP synthase: Using the primordial G. violaceus components as a foundation for building simplified, functional ATP synthases

• Biohybrid energy systems: Incorporating engineered c-rings into artificial membranes for biomimetic energy conversion

The proposal that decreasing c-stoichiometry could increase photosynthetic efficiency presents an intriguing target for synthetic biology approaches. By engineering G. violaceus atpE to form smaller rings, researchers might develop strains with enhanced ATP production efficiency under specific light conditions.

What methodological advances would enhance structural studies of the complete G. violaceus ATP synthase complex?

Several technological advances could significantly enhance structural studies of the complete G. violaceus ATP synthase:

• Advanced cryo-EM techniques: Building on the successful 2.04 Å resolution structure of G. violaceus PSI , similar approaches could resolve the complete ATP synthase

• Nanodiscs and styrene-maleic acid lipid particles (SMALPs): These technologies enable purification of membrane proteins in a more native-like lipid environment

• Time-resolved structural methods: Capturing different conformational states during the catalytic cycle

• Integrative structural biology: Combining cryo-EM, X-ray crystallography, NMR, and computational approaches

Challenges specific to G. violaceus ATP synthase include:

• Obtaining sufficient quantities of the intact complex from native source

• Developing expression systems for co-expression of all subunits

• Maintaining the integrity of the large c₁₅ ring during purification

• Capturing functionally relevant conformational states

The unique evolutionary position of G. violaceus makes structural studies of its ATP synthase particularly valuable for understanding the evolution of this essential bioenergetic complex and the functional significance of its large c-ring stoichiometry.