Recombinant ATP synthase subunit alpha (atpA), partial

What is ATP synthase subunit alpha (atpA) and what role does it play in ATP synthesis?

ATP synthase subunit alpha (atpA) is a key component of the F1 domain of F-type ATP synthase (F₀F₁), which is located in the mitochondrial matrix in eukaryotes or in the cellular membrane of bacteria. As part of the catalytic hexamer (α₃β₃), subunit alpha plays a crucial role in nucleotide binding and the catalytic process of ATP synthesis .

The F1 domain, containing subunit alpha, uses the energy created by the proton electrochemical gradient to phosphorylate ADP to ATP. Specifically, subunit alpha works in conjunction with subunit beta to form three catalytic sites at their interfaces that participate in the rotary mechanism of ATP synthesis. While beta subunits contain the catalytic sites, alpha subunits contribute to nucleotide binding and are essential for the conformational changes that drive ATP synthesis .

What are the structural characteristics of ATP synthase subunit alpha?

ATP synthase subunit alpha exhibits a conserved structural organization across species, though with notable variations in certain organisms such as mycobacteria. The subunit features a nucleotide-binding domain that participates in forming the catalytic interface with beta subunits. The protein contains several conserved motifs involved in nucleotide binding and catalysis .

What expression systems are most effective for producing recombinant atpA?

For recombinant expression of atpA, several expression systems have proven effective, each with distinct advantages depending on research objectives:

• E. coli-based expression systems: The BL21(DE3) strain coupled with pET vectors provides high protein yields for biochemical and structural studies. The expression can be optimized using:

  • Low temperature induction (16-18°C) to enhance proper folding

  • Co-expression with molecular chaperones (GroEL/GroES) to improve solubility

  • Fusion tags (His₆, MBP, or GST) to facilitate purification and enhance solubility

• Cell-free expression systems: These are advantageous for producing proteins that may be toxic to host cells and allow for rapid screening of expression conditions.

• Eukaryotic expression systems: For applications requiring post-translational modifications similar to those in higher organisms, insect cell (Sf9, High Five) or mammalian cell (HEK293, CHO) systems may be preferable .

The choice depends on research requirements, with bacterial systems offering simplicity and high yields, while eukaryotic systems provide more authentic folding and modifications for functional studies.

What purification methods are most effective for recombinant atpA?

Purification of recombinant atpA typically employs a multi-step chromatographic approach:

• Initial capture: Affinity chromatography using nickel-nitrilotriacetic acid (Ni-NTA) for His-tagged proteins or glutathione sepharose for GST-fusion proteins provides high selectivity.

• Intermediate purification: Ion exchange chromatography (typically DEAE or Q-sepharose) can separate the target protein based on charge properties.

• Polishing step: Size exclusion chromatography (Superdex 200 or Sephacryl S-300) ensures high purity and can provide information about the oligomeric state.

• Tag removal: If necessary, the affinity tag can be removed using specific proteases (TEV, PreScission, or thrombin), followed by a reverse affinity step.

Optimal buffer conditions typically include:

• 20-50 mM Tris-HCl or HEPES (pH 7.5-8.0)

• 100-300 mM NaCl to maintain solubility

• 5-10% glycerol for stability

• 1-5 mM DTT or β-mercaptoethanol to prevent oxidation

• Protease inhibitors to minimize degradation

How can I verify the functionality of recombinant atpA in vitro?

Verifying the functionality of recombinant atpA requires multiple complementary approaches:

• Nucleotide binding assays:

  • Fluorescence-based approaches using MANT-ATP or TNP-ATP to measure binding affinities

  • Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of nucleotide binding

  • Surface plasmon resonance (SPR) for real-time binding kinetics

• ATPase activity assays:

  • Coupled enzyme assays (with pyruvate kinase and lactate dehydrogenase) to monitor ADP production

  • Colorimetric assays measuring released inorganic phosphate

  • Luminescence-based ATP detection methods

• Reconstitution experiments:

  • Integration into liposomes with other ATP synthase subunits

  • Proteoliposome-based proton pumping assays using pH-sensitive fluorophores

  • Complementation studies in subunit alpha-deficient bacterial strains

• Structural validation:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Limited proteolysis to assess proper folding

  • Thermal shift assays to evaluate protein stability

These approaches together provide comprehensive validation of recombinant atpA functionality.

What are the challenges in expressing and purifying active recombinant atpA?

Expression and purification of active recombinant atpA presents several challenges:

• Solubility issues: When expressed in isolation from other ATP synthase subunits, atpA may form inclusion bodies or aggregate due to exposed hydrophobic surfaces normally involved in subunit interactions. This can be addressed by:

  • Optimizing expression temperature and inducer concentration

  • Using solubility-enhancing fusion partners (MBP, SUMO, or TrxA)

  • Adding stabilizing agents (glycerol, arginine, or low concentrations of detergents)

• Proper folding: Obtaining correctly folded atpA can be challenging. Strategies include:

  • Co-expression with molecular chaperones

  • Refolding protocols if isolation from inclusion bodies is necessary

  • Co-expression with interaction partners from the ATP synthase complex

• Stability concerns: Isolated atpA may exhibit reduced stability compared to when incorporated in the holoenzyme. This can be mitigated by:

  • Buffer optimization with stabilizing additives

  • Storage at high protein concentrations

  • Addition of nucleotides that may stabilize the protein structure

• Functional validation: Determining if the recombinant protein is functionally equivalent to native atpA requires careful biochemical characterization, especially when expressing partial constructs that might lack important structural elements .

How do post-translational modifications affect the function of atpA?

Post-translational modifications (PTMs) of atpA can significantly impact its function:

• Phosphorylation:

  • Serine/threonine phosphorylation can modulate ATPase activity and interaction with other subunits

  • Methods for studying phosphorylation include mass spectrometry-based phosphoproteomics, phospho-specific antibodies, and in vitro kinase assays

  • Site-directed mutagenesis of potential phosphorylation sites (serine to alanine or aspartate) can help determine functional significance

• Acetylation:

  • Lysine acetylation can affect protein-protein interactions and enzymatic activity

  • Detection methods include anti-acetyllysine antibodies and mass spectrometry

  • Histone deacetylase inhibitors can be used to modulate acetylation levels experimentally

• Oxidative modifications:

  • Cysteine oxidation can impact protein folding and activity

  • Techniques like redox proteomics and biotin-switch assays can identify modified residues

  • Mutational analysis of cysteine residues can assess their functional importance

• Glycosylation:

  • Though less common in mitochondrial proteins, potential glycosylation can be analyzed using glycosidases and lectin-based detection methods

To systematically study PTMs, researchers should compare modifications between recombinant and native proteins using advanced mass spectrometry techniques such as LC-MS/MS with enrichment strategies specific for each modification type .

What structural domains of atpA are most critical for ATP synthesis activity?

Several structural domains in atpA are critical for ATP synthesis activity:

• Nucleotide-binding domain: Contains conserved motifs responsible for ATP/ADP binding and participates in the conformational changes during catalysis. Key features include:

  • P-loop (Walker A motif): Essential for phosphate binding

  • DELSEED region: Important for communication between catalytic and rotary parts of the enzyme

  • Walker B motif: Involved in magnesium coordination

• Interface regions: The regions that interact with β subunits to form the catalytic sites are crucial for enzyme function.

• Interaction sites with the central stalk: These regions communicate conformational changes between the F1 sector and the proton-conducting F0 sector.

• C-terminal domain: In mycobacteria, the unique C-terminal extension (residues 514-549) plays a regulatory role by suppressing ATPase activity through interaction with subunit γ. Deletion of this domain increases ATP hydrolysis activity by approximately 63% .

• N-terminal region: Often involved in the assembly of the F1 complex and stability of the structure.

To identify critical domains experimentally, approaches include:

• Truncation analysis and domain swapping between species

• Alanine scanning mutagenesis of conserved residues

• Hydrogen-deuterium exchange mass spectrometry to identify dynamic regions

• Cross-linking studies to map interaction interfaces

What approaches can be used to study the interaction between atpA and other ATP synthase subunits?

Investigating interactions between atpA and other ATP synthase subunits requires sophisticated approaches:

• Crosslinking coupled with mass spectrometry (XL-MS):

  • Chemical crosslinkers (BS3, DSS, EDC) can capture transient interactions

  • Photo-activated crosslinkers provide spatially precise information

  • Mass spectrometry analysis of crosslinked peptides reveals interaction sites

  • ZeroLength-crosslinkers like EDC can identify residues in direct contact

• Co-immunoprecipitation and pull-down assays:

  • Tag-based pull-downs (His, GST, FLAG) with recombinant proteins

  • Antibody-based immunoprecipitation from native sources

  • Quantitative analysis using SILAC or TMT labeling

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI):

  • Real-time kinetic analysis of protein-protein interactions

  • Determination of association/dissociation constants

  • Effect of nucleotides and other ligands on binding dynamics

• Förster resonance energy transfer (FRET):

  • Site-specific labeling with fluorophore pairs

  • Real-time monitoring of protein interactions in solution

  • Can be combined with single-molecule techniques for detailed mechanistic insights

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

  • Maps interaction interfaces by identifying regions protected from exchange

  • Provides information on conformational changes upon binding

  • Can be performed under various conditions (different nucleotides, pH)

Study of the mycobacterial ATP synthase demonstrated how cross-linking approaches identified interaction between the C-terminal domain of subunit α (residues 521-540) and subunit γ residues 104-109, providing insight into the mechanism of ATPase activity suppression .

How can I investigate the conformational changes in atpA during ATP synthesis?

Investigating conformational changes in atpA during ATP synthesis requires techniques that can capture dynamic structural states:

• Time-resolved cryo-electron microscopy (cryo-EM):

  • Trapping different conformational states using ATP analogs or inhibitors

  • Classification of particle images to identify different conformational states

  • Construction of movies showing the conformational trajectory

  • Resolution of 2-4Å can reveal side chain movements during catalysis

• Single-molecule FRET (smFRET):

  • Strategic placement of donor-acceptor fluorophore pairs at key positions

  • Real-time monitoring of distance changes between labeled sites

  • Can be combined with microfluidics for rapid solution exchange

  • Reveals conformational dynamics at millisecond timescales

• EPR spectroscopy with site-directed spin labeling:

  • Introduction of nitroxide spin labels at specific sites

  • Continuous wave EPR for mobility and accessibility information

  • Double electron-electron resonance (DEER) for precise distance measurements

  • Particularly useful for measuring longer distances (2-8 nm)

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

  • Differential labeling rates identify regions undergoing conformational changes

  • Time-course experiments capture transient states

  • Compatible with large protein complexes and membrane proteins

  • Can be performed under functional conditions

• Molecular dynamics simulations:

  • Integration with experimental data from the above methods

  • Prediction of transition pathways between known conformational states

  • Energetic analysis of conformational changes

  • Testing hypotheses about mechanism before experimental validation

Research on mycobacterial ATP synthase demonstrated that the C-terminal extension of subunit α influences the angular velocity of the power stroke during ATP hydrolysis, highlighting how structural elements can influence conformational dynamics .

What techniques are most effective for studying the rotation mechanism involving atpA in ATP synthase?

Several sophisticated techniques have been developed to study the rotation mechanism of ATP synthase involving atpA:

• Single-molecule rotation assays:

  • Attachment of fluorescent probes (beads, gold nanorods, quantum dots) to the rotor portion

  • Direct visualization of rotation using high-speed cameras or fluorescence microscopy

  • Quantification of step size, dwell times, and angular velocity

  • Analysis of the effect of load, ATP concentration, and inhibitors on rotation

• Fluorescence correlation spectroscopy (FCS):

  • Measurement of nucleotide binding kinetics

  • Determination of the correlation between binding events and rotational steps

  • Assessment of how mutations in atpA affect binding affinities and rotation

• High-speed atomic force microscopy (HS-AFM):

  • Direct visualization of conformational changes in real-time

  • Observation of rotational states without artificial probes

  • Combined with reconstituted systems in lipid bilayers

  • Integration with electrical measurements in nano-chambers

• Magnetic tweezers and optical traps:

  • Application of controlled torque to the rotor

  • Measurement of force generation during rotation

  • Determination of mechanical properties of the motor

  • Assessment of how atpA mutations affect force generation

• Hybrid approaches:

  • Combination of structural methods (cryo-EM, X-ray) with single-molecule techniques

  • Integration of computational simulations with experimental data

  • Correlation of biochemical measurements with mechanical observations

Research on mycobacterial ATP synthase used single-molecule rotation assays with fluorescent beads and gold nanorods to show that the C-terminal extension of subunit α decreased the angular velocity of the power stroke after ATP binding. This demonstrated that structural elements in subunit α can significantly influence the rotational mechanics of ATP synthase .

How can recombinant partial atpA be used as a tool to investigate ATP synthase inhibitors?

Recombinant partial atpA can serve as a valuable tool for investigating ATP synthase inhibitors through several methodological approaches:

• Binding assays for direct inhibitor interactions:

  • Isothermal titration calorimetry (ITC) to measure binding thermodynamics

  • Surface plasmon resonance (SPR) for real-time binding kinetics

  • Microscale thermophoresis (MST) for low sample consumption measurements

  • Fluorescence-based thermal shift assays to detect stabilization upon inhibitor binding

• Competition assays with nucleotides:

  • Fluorescently labeled ATP analogs to monitor displacement by inhibitors

  • Radioligand binding assays with [γ-³²P]ATP or [α-³²P]ATP

  • Analysis of competition kinetics to determine mechanism of inhibition

• Structural studies of inhibitor binding:

  • X-ray crystallography of atpA-inhibitor complexes

  • NMR spectroscopy for mapping binding interfaces

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify conformational changes induced by inhibitors

  • Molecular docking validated by mutagenesis of predicted binding sites

• Functional assays in reconstituted systems:

  • Effect of inhibitors on ATP hydrolysis in reconstituted subcomplexes

  • Impact on conformational dynamics measured by FRET or EPR

  • Influence on rotation mechanics in single-molecule assays

  • Comparison between wild-type and mutant atpA to identify resistance mechanisms

This approach was successfully used to understand the mechanism of the anti-tuberculosis drug bedaquiline (BDQ), which targets mycobacterial ATP synthase. High-resolution structural studies of the complete bacterial F₀F₁ complex helped elucidate how the drug interacts with the enzyme .

What biophysical methods are most suitable for characterizing the nucleotide binding properties of recombinant atpA?

Characterizing nucleotide binding properties of recombinant atpA requires sophisticated biophysical techniques:

• Isothermal Titration Calorimetry (ITC):

  • Provides complete thermodynamic profile (ΔH, ΔS, ΔG)

  • Determines binding stoichiometry and affinity constants

  • Reveals enthalpy-entropy compensation mechanisms

  • Experimental setup:

    • Protein concentration: 10-50 μM

    • Nucleotide concentration: 100-500 μM

    • Buffer conditions: 20-50 mM HEPES/Tris, pH 7.5-8.0, 100-150 mM NaCl, 5-10 mM MgCl₂

• Microscale Thermophoresis (MST):

  • Measures binding in solution with minimal sample consumption

  • Monitors changes in thermophoretic mobility upon ligand binding

  • Compatible with a wide range of buffer conditions

  • Particularly useful for weak interactions (μM to mM range)

• Fluorescence-based approaches:

  • Intrinsic tryptophan fluorescence to monitor conformational changes

  • Fluorescent ATP analogs (MANT-ATP, TNP-ATP) for direct binding measurements

  • Fluorescence anisotropy to detect changes in rotational diffusion upon binding

  • FRET-based assays using strategically placed fluorophores

• Surface Plasmon Resonance (SPR):

  • Real-time kinetic analysis of association/dissociation

  • Determination of kon and koff rates

  • Effect of mutations on binding kinetics

  • Multi-cycle or single-cycle kinetics approaches

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • Chemical shift perturbation to map binding interfaces

  • ¹⁹F-NMR with fluorinated nucleotides for simplified spectra

  • Saturation transfer difference (STD) NMR for epitope mapping

  • Relaxation dispersion experiments for conformational exchange rates

The cross-validation of results from multiple techniques is crucial for robust characterization. For example, fluorescence correlation spectroscopy studies of chimeric ATP synthase containing the mycobacterial C-terminal domain of subunit α showed that binding affinities for ATP or ADP remained unchanged despite decreased ATPase activity, indicating that the suppression mechanism acts downstream of nucleotide binding .

Comparative Structure and Function of ATP Synthase α Subunit Across Species

Effect of C-terminal Modifications on Mycobacterial ATP Synthase Activity

Experimental Conditions for Recombinant atpA Expression and Purification

*SEC: Size Exclusion Chromatography; IEX: Ion Exchange Chromatography

Biophysical Properties of Wild-type and Mutant ATP Synthase α Subunit