Recombinant ATP synthase subunit a (atpB)

What is ATP synthase subunit beta (ATP5B) and what is its role in the ATP synthase complex?

ATP5B (also called ATP5F1B, ATPMB, or ATPSB) is a catalytic subunit of mitochondrial ATP synthase (Complex V). This protein is part of the F1 domain situated in the mitochondrial matrix, while the F0 domain is embedded in the inner mitochondrial membrane. ATP5B plays a crucial role in the catalytic function of ATP synthase, which utilizes the electrochemical gradient of protons across the inner membrane during oxidative phosphorylation to synthesize ATP from ADP and inorganic phosphate. The catalytic β subunits can adopt different conformations and bind to Mg-ADP (βDP), Mg-ATP (βTP), or remain empty (βE) during the catalytic cycle .

How is ATP5B involved in the assembly of the ATP synthase complex?

ATP5B is essential for the proper assembly of the ATP synthase complex. Research indicates that the assembly of complex V occurs in modules, with the F1 domain (containing ATP5B) forming separately from other components. In human mitochondria, studies using clear native polyacrylamide gel electrophoresis (CN-PAGE) have shown that ATP synthase can assemble into a complex with a mass of approximately 550 kDa even in the absence of mitochondrial DNA-encoded subunits. This suggests that ATP5B participates in early assembly stages, before the incorporation of mtDNA-encoded subunits . The assembly process likely involves the formation of the c-ring followed by binding of the F1 domain (which includes ATP5B), then the stator arm, and finally subunits a and A6L .

What expression systems are commonly used for recombinant ATP5B production?

Recombinant ATP5B can be produced using multiple expression systems, each with distinct advantages:

• Bacterial expression (E. coli): A common approach involves transforming E. coli with ATP5F1B expression plasmids after appropriate cDNA preparation and PCR methods. This system typically employs tags such as N-terminal 10xHis-SUMO tag and C-terminal Myc tag, yielding protein with approximately 85%+ purity .

• Yeast expression: Human ATP5F1B (amino acids 48-529) can be expressed in yeast with a 6xHis-tag at the N-terminus, resulting in recombinant full-length mature human ATP5F1B protein with purity greater than 90% and an observed molecular weight of approximately 54 kDa .

The choice between these systems depends on research requirements for protein folding, post-translational modifications, and downstream applications.

What purification methods yield the highest purity recombinant ATP5B?

Obtaining high-purity recombinant ATP5B typically involves a multi-step purification process:

• Affinity chromatography: The primary purification step utilizes affinity tags (His-tag or SUMO-tag) for selective binding to appropriate resins.

• Size exclusion chromatography: This secondary step separates ATP5B from contaminants based on molecular size.

• Ion exchange chromatography: A final polishing step may be employed to remove remaining contaminants based on charge differences.

Using this approach, recombinant ATP5B can be purified to >90% homogeneity as determined by SDS-PAGE analysis . For antibody production or specialized research applications, even higher purity levels may be achieved through additional chromatographic steps or tag removal.

How can recombinant ATP5B be used to study mitochondrial complex V assembly?

Recombinant ATP5B provides valuable tools for investigating the complex assembly process of ATP synthase:

• Tagged ATP5B for interaction studies: Recombinant ATP5B with affinity tags enables pull-down assays to identify interaction partners during various assembly stages. This approach has helped elucidate that ATP synthase assembly in yeast involves separate pathways (F1/Atp9p and Atp6p/Atp8p/stator subunits) that converge at the end stage .

• Fluorescently labeled ATP5B: By introducing ATP5B sequences into fluorescent protein vectors (such as paGFP), researchers can track spatial and temporal dynamics of ATP synthase assembly in living cells. This approach has been used to demonstrate that the ATP synthase complex may be assembled in mitochondria and subsequently transported to the cell surface .

• Mutational analysis: Recombinant ATP5B with specific mutations can reveal the functional importance of different domains in complex assembly. For example, studies have shown that F1 subunit ε plays an indispensable role in holocomplex V assembly and is involved in incorporating subunit c into the rotor structure .

What methods can be used to detect ectopic expression of ATP5B on cellular membranes?

Several methodological approaches can detect ATP5B expression on plasma membranes:

• Immunofluorescence with specific antibodies: Using antibodies that specifically recognize ATP5B, such as monoclonal antibody 4E7 (McAb4E7), researchers can visualize ATP5B localization through immunofluorescence microscopy. This approach has demonstrated that ATP5B is abnormally expressed on the cell surface of non-small cell lung cancer (NSCLC) cells but not small cell lung cancer (SCLC) cells .

• Flow cytometry: This technique provides quantitative analysis of surface-expressed ATP5B. Flow cytometric analysis with specific antibodies has confirmed the localization of ATP5B on the surface of A549 lung adenocarcinoma cells .

• Plasma membrane fractionation and proteomic analysis: By isolating plasma membrane fractions and performing proteomic analysis, researchers have detected ATP5B and other mitochondrial proteins in the plasma membrane. This approach revealed that eATP synthase might reach the plasma membrane via mitochondria-dependent transportation .

How does ectopic ATP5B on plasma membranes contribute to cancer pathophysiology?

The presence of ATP5B on plasma membranes (eATP synthase) has significant implications for cancer research:

• Potential biomarker: Immunohistochemistry studies have shown that ectopic ATP5B is predominantly expressed in non-small cell lung cancer (NSCLC) tumor cell membranes, with expression rates of 71.88% in lung adenocarcinoma and 66.67% in squamous carcinoma, compared to only 25.81% in adjacent non-tumorous lung tissues. This makes ATP5B a potential diagnostic biomarker for NSCLC .

• Therapeutic target: Monoclonal antibodies targeting ectopic ATP5B, such as McAb4E7, have demonstrated inhibitory effects on cancer cell proliferation. This suggests that abnormally expressed ATP5B on cell surfaces might serve as a tumor-associated antigen for immunotherapy in NSCLC .

• Metabolic adaptation: Surface ATP5B may contribute to cancer cell energy metabolism by generating ATP in the microenvironment, potentially supporting cancer cell survival under stressful conditions. The association of ATP synthase with other metabolic enzymes on the plasma membrane suggests coordinated metabolic activity outside mitochondria .

What computational methods are available for predicting ATP-binding residues in ATP5B?

Several computational approaches have been developed to predict ATP-binding residues:

These computational tools help identify potential ATP-binding residues, guiding experimental design for mutagenesis studies and structural investigations of ATP5B.

What are the best approaches for studying the conformational changes of ATP5B during catalysis?

Studying ATP5B conformational dynamics requires sophisticated methodological approaches:

• Cryo-electron microscopy (cryo-EM): This technique has revealed the arrangement of subunits in intact mammalian mitochondrial ATP synthase, showing that the minimal contact between the a-subunit and c8-ring limits protein-protein interactions that would impede rotation necessary for catalysis . For ATP5B specifically, cryo-EM can capture different conformational states (βDP, βTP, or βE) during the catalytic cycle.

• Single-molecule FRET (Förster Resonance Energy Transfer): By labeling ATP5B at specific sites with fluorescent dyes, researchers can monitor real-time conformational changes during catalysis at the single-molecule level.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This approach can map regions of ATP5B that undergo conformational changes during catalysis by measuring the rate of hydrogen-deuterium exchange in different functional states.

• Molecular dynamics simulations: Computational methods can model ATP5B conformational changes based on structural data, providing insights into the molecular mechanisms of catalysis that may be difficult to capture experimentally.

How can researchers investigate the interaction between ATP5B and other subunits of the ATP synthase complex?

Several methods are effective for studying ATP5B interactions with other complex V components:

• Co-immunoprecipitation with tagged recombinant ATP5B: Using affinity-tagged recombinant ATP5B, researchers can pull down interacting partners and identify them through mass spectrometry. This approach has helped establish the assembly sequence of ATP synthase subunits .

• Crosslinking mass spectrometry: Chemical crosslinking followed by mass spectrometry analysis can identify physical contacts between ATP5B and other subunits, providing spatial constraints for structural modeling.

• Yeast two-hybrid or mammalian two-hybrid systems: These methods can verify direct interactions between ATP5B and other subunits in a cellular context.

• Proximity labeling approaches (BioID or APEX): By fusing ATP5B with promiscuous biotin ligases, researchers can identify proteins that are in close proximity to ATP5B in living cells, revealing the spatial organization of the complex.

These methodologies provide complementary information about ATP5B interactions, helping researchers understand both stable and transient associations within the ATP synthase complex.

What are the common challenges in expressing functional recombinant ATP5B and how can they be addressed?

Researchers often encounter several challenges when producing recombinant ATP5B:

• Solubility issues: ATP5B may form inclusion bodies in bacterial expression systems. This can be addressed by:

  • Optimizing growth temperature (typically lowering to 16-18°C)

  • Using solubility-enhancing tags like SUMO or MBP

  • Employing specific E. coli strains designed for improved protein folding

  • Adding solubilizing agents during purification

• Proper folding: As a complex protein normally assembled within mitochondria, ATP5B may not fold correctly when expressed recombinantly. Solutions include:

  • Expression in eukaryotic systems like yeast for better folding machinery

  • Co-expression with chaperone proteins

  • Refolding protocols if recovery from inclusion bodies is necessary

• Functionality assessment: Verifying that recombinant ATP5B retains its native activity is critical. This can be accomplished through:

  • ATPase activity assays

  • Binding studies with known interaction partners

  • Structural analysis to confirm proper folding

How can the purity and quality of recombinant ATP5B be assessed for specific research applications?

Thorough quality control of recombinant ATP5B involves multiple analytical methods:

• Purity assessment:

  • SDS-PAGE with densitometry analysis (target >90% purity)

  • Size exclusion chromatography to detect aggregates or degradation products

  • Mass spectrometry to confirm protein identity and detect contaminants

• Structural integrity:

  • Circular dichroism (CD) spectroscopy to verify secondary structure

  • Thermal shift assays to assess protein stability

  • Limited proteolysis to confirm proper folding

• Functional validation:

  • ATP binding assays

  • ATPase activity measurements

  • Interaction studies with known binding partners

For specific applications, additional quality controls may be necessary. For instance, if using recombinant ATP5B for antibody production, testing for endotoxin contamination is essential, while structural studies may require additional verification of homogeneity through dynamic light scattering.