Recombinant Anabaena variabilis ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Anabaena variabilis?

ATP synthase subunit c (atpE) in Anabaena variabilis is a small, highly hydrophobic membrane protein consisting of 81 amino acids with the sequence: MDPLVSAASVLAAALAVGLAAIGPGIGQGNAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALVLLFANPFA . This protein forms part of the F0 sector of ATP synthase, where multiple c-subunits assemble into a ring structure (c-ring) embedded within the membrane. The primary function of the c-subunit is to participate in proton translocation across the membrane, which drives the rotation of the c-ring.

The c-subunit typically contains two transmembrane helices connected by a short loop, with a critical glutamate residue that undergoes reversible protonation/deprotonation during the proton translocation cycle. This glutamate residue is conserved across species and is essential for function. The c-ring structure contains 8-15 subunits depending on the species, and it interfaces with the a-subunit to form the proton translocation pathway.

The rotation of the c-ring is mechanically coupled to the F1 sector of ATP synthase, inducing conformational changes in the catalytic sites that lead to ATP synthesis. This coupling represents one of nature's most elegant molecular machines for energy conversion in biological systems.

How does the c-subunit contribute to ATP synthesis?

The c-subunit plays a pivotal role in the energy conversion mechanism of ATP synthase through several key functions:

• It forms a cylindrical ring (c-ring) in the membrane composed of multiple identical subunits, with the exact number varying by species.

• Each c-subunit contains a conserved carboxyl group (usually glutamate) that serves as the proton-binding site.

• The c-ring rotates relative to the a-subunit during proton translocation.

• This rotation is directly coupled to the γ-subunit in the F1 sector, driving conformational changes that catalyze ATP synthesis.

During ATP synthesis, protons move down their electrochemical gradient through a channel at the interface between the a-subunit and c-ring . Each proton binds to the key glutamate residue on a c-subunit, causing protonation and a subtle conformational change. As the c-ring rotates, the protonated glutamate moves through the hydrophobic membrane environment until it reaches another interface with the a-subunit, where the proton is released to the opposite side of the membrane .

Ion translocation through the F0 sector drives rotation of the cylindrical ring of c-subunits that is coupled to rotation of the γ-subunit within the α3β3 hexamer of F1, a rotation that ultimately drives synthesis of ATP . This elegant mechanism effectively converts the energy of the proton gradient into mechanical rotation and then into chemical energy stored in ATP.

What are the key conserved residues in the atpE protein across species?

Despite considerable sequence variability in the atpE protein across species, certain key residues are highly conserved due to their critical roles in function:

The most critical conserved residue is a carboxyl-containing amino acid (typically glutamate) that serves as the proton-binding site . In different organisms, this corresponds to different positions as shown in the table above. This glutamate provides the main stabilizing interaction for the H+/Na+ ion within the c-ring structure .

Additional conserved residues include those that provide secondary stabilization for the bound proton. For example, in M. tuberculosis, Asp28 (corresponding to Gln32 in I. tartaricus and Gln29 in S. platensis) contributes to stabilizing the proton binding site .

The functional importance of these conserved residues is demonstrated by mutation studies. For instance, the E56D mutation in Bacillus PS3 reduces ATP synthesis activity but doesn't eliminate it completely, while the more drastic E56Q mutation abolishes activity entirely . This shows that both the presence of a carboxyl group and its precise positioning are critical for proper function.

What expression systems are commonly used for producing recombinant atpE?

The expression and purification of recombinant atpE protein typically involves several carefully optimized steps:

• Expression system selection:

  • E. coli is the most commonly used expression host for atpE, as evidenced by the recombinant Anabaena variabilis atpE product description .

  • BL21(DE3) or similar strains are frequently preferred for membrane protein expression.

  • Temperature-controlled expression (typically at 25-30°C rather than 37°C) often improves proper folding.

• Vector design considerations:

  • Vectors with controllable promoters (T7, tac, or arabinose-inducible)

  • Addition of affinity tags (His-tag is commonly used, as seen in the commercial product)

  • Fusion partners to improve solubility (e.g., MBP, SUMO, or Trx)

  • Signal sequences for membrane targeting if needed

• Optimization parameters:

  • Induction conditions (inducer concentration, temperature, duration)

  • Media composition (often enriched for membrane proteins)

  • Codon optimization for the expression host

  • Cell lysis and membrane fraction isolation methods

For the Anabaena variabilis atpE specifically, the recombinant protein was expressed in E. coli with an N-terminal His-tag, covering the full-length protein (81 amino acids) . The product is supplied as a lyophilized powder in Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain stability during storage .

After expression, membrane proteins like atpE require specialized solubilization and purification approaches using appropriate detergents, followed by quality assessment through techniques such as SDS-PAGE to ensure purity (typically >90% for research applications) .

How do you assess the purity and functionality of recombinant atpE protein?

Comprehensive assessment of recombinant atpE requires both purity verification and functional testing:

Purity Assessment:

• SDS-PAGE analysis: The primary method to verify protein purity, with >90% purity typically considered appropriate for research applications .

• Western blotting: Using anti-His antibodies (for tagged proteins) or specific anti-atpE antibodies.

• Mass spectrometry: For accurate molecular weight determination and sequence verification.

• Size exclusion chromatography: To detect aggregates or oligomeric states.

Functional Assessment:

• Structural integrity tests:

  • Circular dichroism spectroscopy to verify secondary structure content (primarily α-helical)

  • Thermal stability assays to ensure proper folding

• Binding assays:

  • Interaction with known inhibitors (e.g., DCCD or oligomycin)

  • Binding to other ATP synthase subunits

  • pH-dependent conformational changes

• Reconstitution studies:

  • Incorporation into liposomes with other ATP synthase components

  • Proton translocation assays using pH-sensitive fluorescent dyes

  • ATP synthesis/hydrolysis activity when incorporated into a functional complex

For the recombinant Anabaena variabilis atpE, the product is prepared as a lyophilized powder with 6% trehalose and supplied in a Tris/PBS-based buffer at pH 8.0 . For reconstitution, it's recommended to dissolve the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol for long-term storage . Proper handling includes avoiding repeated freeze-thaw cycles and storing working aliquots at 4°C for no more than one week .

How do mutations in specific residues of atpE affect proton translocation and ATP synthesis?

Mutations in key residues of atpE have revealed important insights about the mechanism of proton translocation and ATP synthesis:

Conserved Glutamate Mutations:

The E56D mutation in Bacillus PS3 (involving substitution of glutamate with aspartate) significantly decreases but does not completely eliminate ATP synthesis activity . This partial retention of activity indicates that while the carboxyl group is essential for function, the shorter side chain of aspartate affects the efficiency of proton binding and release . In contrast, more drastic mutations like E56Q (glutamate to glutamine) completely abolish ATP synthesis activity, confirming that a protonatable carboxyl group is absolutely essential for function .

Position-Dependent Effects:

In Mycobacterium tuberculosis, six distinct mutations have been identified in the c-subunit of TMC207-resistant strains: Asp28→Gly, Asp28→Ala, Leu59→Val, Glu61→Asp, Ala63→Pro, and Ile66→Met . These mutations affect both drug binding and the functional properties of the c-subunit, highlighting how subtle changes in protein structure can have significant consequences for both activity and inhibitor interactions.

Cooperative Effects Among c-Subunits:

One of the most intriguing findings is that when double E56D mutations are introduced into the c-ring of Bacillus PS3 ATP synthase, the activity decreases more dramatically as the distance between the mutated c-subunits increases . This reveals functional cooperation among c-subunits during the rotation cycle. Specifically:

Molecular dynamics simulations suggest that mutations affect the duration of proton uptake steps, and when mutations are introduced in distant c-subunits, the waiting time for proton uptake cannot be shared between them, explaining the observed activity patterns .

These findings collectively demonstrate that proton translocation involves cooperation among multiple c-subunits and that the precise positioning of the proton-binding residues is critical for optimal ATP synthase function.

What techniques can be used to study the rotation of the c-ring during ATP synthesis?

Investigating the rotation of the c-ring requires sophisticated techniques that can detect molecular motion at the nanoscale:

Single-Molecule Fluorescence Techniques:

• Single-molecule FRET (Fluorescence Resonance Energy Transfer):

  • Attachment of fluorescent donor and acceptor dyes to specific positions

  • Detection of distance changes during rotation

  • Real-time observation of rotational steps and their kinetics

• Fluorescent probe attachment:

  • Labeling of c-ring with fluorescent actin filaments or gold nanorods

  • Visualization using fluorescence or dark-field microscopy

  • Quantitative measurement of rotational parameters (speed, step size, torque)

Biophysical and Biochemical Approaches:

• Site-specific cross-linking:

  • Introduction of cysteine residues at strategic positions

  • Formation of disulfide bridges to trap rotational intermediates

  • Analysis of cross-linking patterns to infer rotation mechanism

• Genetic approaches with engineered c-rings:

  • Construction of genetically fused single-chain c-rings

  • Introduction of specific mutations at defined positions

  • Analysis of functional consequences to infer rotational dynamics

Research on Bacillus PS3 ATP synthase demonstrated this approach by creating a fusion construct in which 10 copies of the c-subunit were genetically linked to form a single polypeptide . This allowed the researchers to introduce mutations at specific positions and study how the pattern of mutations affected enzyme activity, providing insights into the cooperative nature of c-ring rotation .

Computational Methods:

Molecular dynamics simulations provide valuable insights into the rotation mechanism, particularly when integrated with experimental data. These simulations can reveal energy barriers in the rotation process, the pathway of proton movement, and the coupling between proton translocation and c-ring rotation .

These combined approaches have demonstrated that the c-ring rotates in discrete steps, with each step corresponding to the translocation of one proton, and that there is functional cooperation among adjacent c-subunits during this process.

How can researchers investigate the cooperation among c-subunits in the ATP synthase complex?

Investigating cooperation among c-subunits requires specialized approaches that can differentiate the roles of individual subunits within the c-ring:

Genetically Engineered c-Rings:

A breakthrough approach involves the creation of genetically fused c-rings, where multiple c-subunits are connected into a single polypeptide chain . This strategy, successfully applied with Bacillus PS3 ATP synthase, allows researchers to:

• Introduce specific mutations (e.g., E56D) at precisely defined positions within the c-ring

• Create various combinations of wild-type and mutated c-subunits

• Analyze how the pattern and positioning of mutations affect enzyme activity

Research using this approach revealed that ATP synthesis activity decreases progressively with the number of mutations and, importantly, with increasing distance between mutated sites . This pattern strongly indicates functional cooperation among c-subunits.

Proton Transfer-Coupled Molecular Simulations:

Computational approaches provide insights into the molecular basis of cooperation:

• Simulation of proton movement through multiple c-subunits

• Analysis of how mutations affect proton uptake and release kinetics

• Correlation of simulation results with experimental activity data

Simulations have revealed that up to three deprotonated carboxyl residues can face the a-subunit simultaneously, and the waiting time for proton uptake can be shared between these subunits . When mutations are introduced at sites that are far apart, this time-sharing mechanism becomes less effective, explaining the observed activity patterns in the mutant enzymes .

Functional Measurements:

The degree of cooperation can be quantified through:

• ATP synthesis assays: Measurement of ATP production rates under defined conditions

• Proton pump activity: Monitoring proton movement in the ATP hydrolysis direction

• Rotation assays: Direct measurement of c-ring rotation in mutant enzymes

What are the implications of atpE mutations for drug resistance in pathogenic bacteria?

Mutations in the atpE gene have significant implications for drug resistance, particularly against compounds that target the ATP synthase:

Diarylquinoline Resistance:

TMC207 (Bedaquiline) is an antituberculous drug that very efficiently inhibits the ATP synthase of mycobacteria such as Mycobacterium tuberculosis . Research has identified six distinct mutations in the c-subunit that confer resistance to TMC207:

These mutations were identified by selecting in vitro TMC207-resistant mutants from M. tuberculosis and diverse atypical mycobacteria . The mutations were then studied by evaluating the levels of resistance they confer in the selected clones and using isogenic complementation systems .

Resistance Mechanisms:

The identified mutations affect the binding of TMC207 to the c-subunit through various mechanisms:

• Direct disruption of drug binding sites

• Alteration of proton binding and translocation pathways

• Changes in c-ring structure and dynamics

• Modification of interactions between c-subunits

These findings are particularly important because they help understand the molecular basis of resistance to ATP synthase inhibitors and can guide the development of new drugs or treatment strategies that can overcome resistance.

Fitness Costs and Compensation:

Many atpE mutations that confer drug resistance also reduce ATP synthase efficiency, potentially resulting in reduced bacterial growth rates or virulence. This fitness cost may limit the spread of resistant strains in the absence of drug pressure. Understanding these fitness costs and potential compensatory mutations is critical for predicting the evolution of resistance in clinical settings.

How do structural differences in atpE across species impact its functional properties and inhibitor binding?

Structural differences in atpE across species have significant implications for both function and inhibitor interactions:

Sequence and Structural Variation:

Despite functional conservation, atpE shows considerable sequence variation across species. For example, Anabaena variabilis atpE is 81 amino acids long with the sequence: MDPLVSAASVLAAALAVGLAAIGPGIGQGNAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALVLLFANPFA . This differs from other organisms while maintaining key functional residues.

Functional Implications:

Species-specific differences in atpE lead to variations in:

• Proton/sodium selectivity: Some species use Na+ instead of H+ for ATP synthesis

• pH optima and thermal stability: Reflecting adaptation to different environments

• Coupling efficiency: The ratio of protons translocated per ATP synthesized

• Rotation mechanics: Affecting the torque generation and step size

Inhibitor Binding and Selectivity:

The structural differences between species are particularly relevant for inhibitor binding:

• Species-specific binding pockets: TMC207 (Bedaquiline) selectively inhibits mycobacterial ATP synthase but not human ATP synthase

• Variable sensitivity to inhibitors: Mutations that confer resistance in one species may not have the same effect in others

• Potential for selective targeting: Understanding structural differences enables the design of species-specific inhibitors

The detailed study of mutations in mycobacterial ATP synthase has shown how specific amino acid changes can affect drug binding while maintaining essential functionality . This information is valuable for developing new antibiotics that target ATP synthase in pathogenic bacteria without affecting the human enzyme.

These structural and functional differences across species provide opportunities for the development of selective therapeutic agents that target specific pathogens while minimizing effects on human cells or beneficial microbiota.

What protocols are recommended for site-directed mutagenesis of the atpE gene?

Site-directed mutagenesis of the atpE gene requires specific considerations due to its small size and hydrophobic nature:

PCR-Based Methods:

• QuikChange Method:

  • Design complementary primers containing the desired mutation

  • Perform PCR with high-fidelity polymerase

  • Digest template DNA with DpnI to remove the unmutated template

  • Transform into competent cells

• Overlap Extension PCR:

  • Design two sets of primers: external primers and internal primers containing the mutation

  • Perform two separate PCR reactions to generate overlapping fragments

  • Use the overlapping fragments as templates for a third PCR with external primers

  • Clone the final product into an expression vector

The atpE gene of mycobacteria has been successfully amplified using degenerate primers atpBS and atpFAS, as described in previous research . For Anabaena variabilis atpE, similar approaches could be applied with primers designed based on its 243 bp sequence.

Cloning Considerations:

When working with atpE, several vector systems have proven effective:

• pMOSBlue vector: Used for initial cloning of PCR products

• pLYG204.zeo: Used for expression of atpE genes from different species

• Expression vectors with appropriate tags: Adding His-tags facilitates purification

Previous research successfully cloned and expressed atpE genes from M. smegmatis and M. tuberculosis by first inserting them into the pMOSBlue vector and then transferring them to the pLYG204.zeo plasmid .

Verification Methods:

After mutagenesis, thorough verification is essential:

• Complete DNA sequencing of the entire gene to confirm the desired mutation and absence of unwanted changes

• Restriction enzyme analysis if the mutation creates or eliminates a restriction site

• Functional complementation to verify the biological effect of the mutation

For studying functional effects, an isogenic complementation system in M. smegmatis has been used to evaluate mutations in the atpE gene from M. tuberculosis , demonstrating the importance of appropriate model systems for functional validation.

These protocols allow precise engineering of atpE for structure-function studies, investigation of proton translocation mechanisms, and analysis of drug resistance.

How can researchers effectively reconstitute recombinant atpE into liposomes for functional studies?

Reconstitution of recombinant atpE into liposomes for functional studies involves several critical steps:

Preparation of Purified atpE Protein:

• Express the protein with an affinity tag (typically His-tag) as described for the Anabaena variabilis atpE product

• Purify using affinity chromatography in the presence of appropriate detergents

• Verify purity by SDS-PAGE (>90% purity is recommended)

• Concentrate while maintaining solubility

Liposome Preparation:

• Select appropriate lipids:

  • E. coli polar lipid extract for bacterial proteins

  • DOPC:DOPE:DOPG mixtures for general reconstitution

  • Consider matching lipid composition to the native membrane when possible

• Prepare liposomes:

  • Dissolve lipids in chloroform

  • Dry under nitrogen gas and then under vacuum

  • Hydrate with reconstitution buffer

  • Sonicate or extrude to form uniform liposomes

Reconstitution Methods:

• Detergent-mediated reconstitution:

  • Mix protein and liposomes at the desired protein:lipid ratio

  • Add detergent to destabilize liposomes

  • Remove detergent by Bio-Beads, dialysis, or gel filtration

• Optimization parameters:

  Parameter	Range	Notes
  Protein:lipid ratio	1:50 to 1:200 (w/w)	Higher protein may cause aggregation
  Detergent concentration	0.5-2%	Must exceed CMC but avoid excess
  Buffer pH	6.8-8.0	Match physiological conditions
  Temperature	4-25°C	Lower for stability, higher for efficiency
  Salt concentration	50-200 mM	Affects proteoliposome stability

Functional Verification:

• Proton pumping assay:

  • Use pH-sensitive dyes (ACMA or pyranine)

  • Monitor pH changes upon energization

• ATP synthesis assay:

  • When reconstituted with complete ATP synthase

  • Measure ATP production in response to proton gradient

• Orientation analysis:

  • Use proteolytic digestion of exposed regions

  • Assess the symmetry of protein incorporation

This methodology enables functional studies of atpE in a membrane environment that mimics its native condition, allowing investigation of proton translocation, inhibitor binding, and interactions with other ATP synthase subunits.

What spectroscopic techniques are most informative for studying conformational changes in atpE?

Several spectroscopic techniques provide valuable insights into atpE conformational dynamics:

Circular Dichroism (CD) Spectroscopy:

CD spectroscopy is particularly useful for monitoring the predominantly α-helical secondary structure of atpE:

• Far-UV CD (190-250 nm): Quantifies α-helical content and detects changes in secondary structure

• Near-UV CD (250-350 nm): Probes tertiary structure through aromatic amino acid environments

• Applications:

  • Monitoring pH-dependent conformational changes

  • Detecting structural perturbations upon inhibitor binding

  • Assessing protein stability under various conditions

Fluorescence Spectroscopy:

Fluorescence approaches provide sensitive detection of local conformational changes:

• Intrinsic fluorescence:

  • Monitoring tryptophan or tyrosine residues if present

  • Detecting changes in the microenvironment of aromatic residues

• Extrinsic fluorescence:

  • Site-directed fluorescence labeling at introduced cysteine residues

  • FRET pairs to measure specific distances within the protein

  • Environment-sensitive probes to detect conformational transitions

Vibrational Spectroscopy:

These techniques are particularly valuable for monitoring protonation states:

• FTIR (Fourier Transform Infrared) Spectroscopy:

  • Monitors protonation states of carboxyl groups (critical for glutamate residues)

  • Detects hydrogen bonding changes

  • Distinguishes different conformational states

• Raman Spectroscopy:

  • Complementary to FTIR with less interference from water

  • Can be performed on reconstituted proteoliposomes

  • Provides information about side chain orientations

Advanced NMR Approaches:

NMR spectroscopy offers atomic-level insights into structure and dynamics:

These techniques, often used in combination, provide complementary information about atpE structural dynamics, protonation-dependent conformational changes, and interactions with inhibitors or other ATP synthase subunits. The choice of technique depends on the specific question being addressed and the available protein sample.

How can molecular dynamics simulations be used to investigate proton movement through the c-ring?

Molecular dynamics (MD) simulations have become invaluable tools for understanding the molecular mechanisms of proton movement through the ATP synthase c-ring:

Simulation Setup:

• System preparation:

  • Full atomistic model of c-ring embedded in lipid bilayer

  • Explicit solvent molecules and ions

  • Appropriate force field selection (CHARMM36 or AMBER for membrane proteins)

• Protonation state assignment:

  • pKa calculations for key titratable residues

  • Definition of protonation states for glutamate residues

  • Consideration of multiple protonation scenarios

Enhanced Sampling Techniques:

Standard MD simulations are often insufficient to capture proton transfer events that occur on longer timescales. Enhanced techniques include:

• Constant pH molecular dynamics (CpHMD):

  • Allows protonation states to change during simulation

  • Couples protonation events to conformational changes

• Metadynamics or umbrella sampling:

  • Explores energy barriers for proton transfer

  • Reconstructs free energy profiles for proton movement

Proton Transfer Specific Methods:

Specialized approaches for modeling proton movement include:

• MS-EVB (Multi-State Empirical Valence Bond):

  • Models reactive events like proton transfer

  • Allows bond breaking and formation during simulation

• QM/MM approaches:

  • Quantum mechanical treatment of proton transfer region

  • Classical treatment of surrounding protein and environment

Research on the Bacillus PS3 ATP synthase demonstrated how MD simulations can reproduce and explain experimental observations . Simulations showed that when E56D mutations were introduced at different positions in the c-ring, the prolonged duration times for proton uptake could be shared between adjacent mutated subunits . As the distance between mutations increased, this sharing became less effective, explaining the progressive decrease in activity observed experimentally .

These simulations revealed that at least three c-subunits at the a/c interface cooperate during c-ring rotation, consistent with the presence of multiple deprotonated carboxyl residues facing the a-subunit . This computational insight provided a molecular explanation for the mysterious cooperation observed in biochemical assays.

What approaches can be used to study interactions between atpE and other subunits of the ATP synthase complex?

Studying interactions between atpE and other ATP synthase subunits requires specialized techniques for membrane protein complexes:

Crosslinking Approaches:

• Chemical crosslinking:

  • Use of bifunctional reagents with varying spacer lengths

  • Mass spectrometry analysis of crosslinked peptides

  • Mapping of interaction interfaces at amino acid resolution

• Photo-crosslinking:

  • Incorporation of photo-activatable amino acids at specific positions

  • Site-specific probing of interaction interfaces

  • High spatial and temporal resolution

Genetic Methods:

• Second-site suppressor analysis:

  • Introduction of mutations in atpE

  • Identification of compensatory mutations in other subunits

  • Reveals functional interactions between subunits

• Construction of fusion proteins:

  • Direct linking of interacting subunits (as demonstrated with the c10 fusion protein)

  • Analysis of fusion protein function

  • Constrains interactions in defined orientations

Biochemical Techniques:

• Co-purification assays:

  • Affinity tags on atpE or other subunits

  • Analysis of co-purifying components

  • Identification of stable interactions

• Blue native PAGE:

  • Separation of intact complexes

  • Western blotting for subunit composition

  • Assessment of complex stability

Structural Methods:

• Cryo-electron microscopy:

  • Structure determination of the entire ATP synthase complex

  • Localization of subunit interfaces at near-atomic resolution

  • Visualization of different functional states

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Identification of protected regions at interfaces

  • Detection of conformational changes upon binding

  • Dynamic information complementary to static structures

Integrated Approaches:

Research has demonstrated that these approaches can reveal critical interactions between the c-ring and a-subunit at the proton channel, as well as interactions with the γ and ε subunits that couple c-ring rotation to conformational changes in the F1 sector .