Recombinant Aneura mirabilis ATP synthase subunit b, chloroplastic (atpF)

What is Aneura mirabilis and why is its ATP synthase of special interest?

Aneura mirabilis (formerly known as Cryptothallus mirabilis) is an extraordinary parasitic liverwort that lacks chlorophyll and has plastids that do not differentiate into chloroplasts . Despite being non-photosynthetic, this mycoheterotrophic plant retains plastid-encoded genes for ATP synthase subunits, including atpF, which encodes the b subunit of the ATP synthase complex . This retention makes A. mirabilis an excellent model system for studying the evolution and adaptation of chloroplast genes in non-photosynthetic plants and provides insight into the essential non-photosynthetic functions of ATP synthase.

What is the function of ATP synthase subunit b (atpF) in plastids?

ATP synthase subunit b (atpF) is a critical component of the "stator stalk" (or "peripheral stalk") in the ATP synthase complex. In photosynthetic organisms, this complex couples proton translocation across the thylakoid membrane with ATP synthesis. The stator stalk, composed of subunits b₂δ in bacteria or equivalent structures in eukaryotes, anchors the stationary parts of the complex together and must withstand significant elastic strain (equivalent to about 50 kJ/mol) during rotary catalysis . While most components of ATP synthase are involved in energy production associated with photosynthesis, in non-photosynthetic plastids like those in A. mirabilis, the retention of ATP synthase components suggests alternative or essential functions that extend beyond photosynthesis .

What expression systems are most suitable for recombinant production of atpF?

Based on successful expression of similar ATP synthase components, Escherichia coli BL21(DE3) is the most widely used expression system for recombinant production of plastid ATP synthase subunits . For the expression of A. mirabilis atpF, the following approaches have proven effective:

When expressing atpF, it's crucial to optimize induction conditions (temperature, IPTG concentration) as membrane-associated proteins can be challenging to express in soluble form. For A. mirabilis atpF specifically, expression at lower temperatures (16-18°C) following induction with 0.1-0.5 mM IPTG has shown improved solubility compared to standard conditions .

What is the most effective purification strategy for recombinant A. mirabilis atpF?

Purification of recombinant A. mirabilis atpF typically employs immobilized metal affinity chromatography (IMAC) when the protein contains a histidine tag . The recommended purification protocol involves:

• Cell lysis using sonication or French press in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and protease inhibitors

• Clarification of lysate by centrifugation (20,000 × g, 30 minutes, 4°C)

• Binding to Ni-NTA resin in the presence of 10-20 mM imidazole to reduce non-specific binding

• Washing with increasing imidazole concentrations (20-50 mM)

• Elution with 250-300 mM imidazole

• Buffer exchange via dialysis or size exclusion chromatography to remove imidazole

For improved stability during purification and storage, inclusion of 6% trehalose in the final buffer formulation has been shown to significantly enhance protein stability . The purified protein should be stored at -20°C/-80°C, with aliquoting recommended to avoid repeated freeze-thaw cycles .

What are the key structural and biochemical properties of A. mirabilis atpF?

While specific structural data for A. mirabilis atpF is limited, analysis of homologous ATP synthase b subunits suggests the following characteristics:

• Predicted structure: N-terminal transmembrane helix followed by an extended cytoplasmic (or stromal) helix

• Oligomeric state: Typically forms dimers (b₂) in the context of the ATP synthase complex

• Stability: In isolation, moderate stability that can be enhanced with appropriate buffer conditions

• Interactions: Forms strong interactions with the δ subunit and other components of the ATP synthase complex

Understanding these properties is essential for experimental design when working with the recombinant protein, particularly for functional and structural studies.

How can recombinant A. mirabilis atpF be used for functional studies?

Recombinant A. mirabilis atpF enables several experimental approaches for functional characterization:

• Reconstitution studies: Purified atpF can be combined with other ATP synthase subunits to reconstruct partial or complete complexes in vitro, allowing assessment of assembly dynamics and functional properties .

• Protein-protein interaction studies: Techniques such as pull-down assays, surface plasmon resonance, or isothermal titration calorimetry can be used to quantitatively assess interactions between atpF and other ATP synthase components .

• Comparative biochemistry: Functional properties of A. mirabilis atpF can be compared with homologs from photosynthetic organisms to identify adaptations specific to non-photosynthetic plastids .

• Structural biology: Purified protein can be used for crystallization trials or cryo-electron microscopy studies to determine high-resolution structures .

These approaches provide complementary information about the role and properties of atpF in the context of ATP synthase function in non-photosynthetic plastids.

What challenges might arise when working with recombinant A. mirabilis atpF?

Researchers working with recombinant A. mirabilis atpF should be prepared for several common challenges:

• Solubility issues: As a membrane-associated protein, atpF may form inclusion bodies during expression. This can be addressed by:

  • Optimizing expression conditions (lower temperature, reduced inducer concentration)

  • Using solubility-enhancing fusion partners (such as MBP or SUMO)

  • Incorporating mild detergents in lysis and purification buffers

• Protein stability: Purified atpF may exhibit limited stability in solution. Consider:

  • Addition of stabilizing agents (glycerol, trehalose) to storage buffers

  • Aliquoting and storing at -80°C to prevent freeze-thaw degradation

  • Performing functional studies immediately after purification

• Functional assessment: As part of a multi-subunit complex, isolated atpF may not display measurable enzymatic activity on its own, necessitating more complex reconstitution approaches .

How has atpF evolved in A. mirabilis given its non-photosynthetic lifestyle?

The evolution of atpF in A. mirabilis represents a fascinating case study in gene retention and adaptation following the loss of photosynthesis. While photosynthetic organisms maintain ATP synthase genes under strong selective pressure , non-photosynthetic organisms often lose chloroplast genes that are specifically associated with photosynthesis.

• Essential non-photosynthetic functions: The ATP synthase complex likely performs critical functions beyond photosynthesis, which is why these genes are retained even after the loss of photosynthetic capacity .

• Relaxed selective pressure: Comparative sequence analysis may reveal evidence of relaxed selection in certain regions of the protein, particularly those that might have been important specifically for photosynthesis-related functions .

• Functional constraints: Despite the loss of photosynthesis, structural elements of atpF that are crucial for ATP synthase assembly and function would remain under purifying selection .

Researchers can investigate these evolutionary patterns through comparative genomics and molecular evolution analyses, comparing sequence conservation patterns between photosynthetic and non-photosynthetic relatives.

What insights can recombinant A. mirabilis atpF provide about ATP synthase assembly in non-photosynthetic plastids?

Recombinant A. mirabilis atpF serves as a valuable tool for investigating ATP synthase assembly in non-photosynthetic plastids:

• Interaction specificity: By comparing the binding affinity and specificity of A. mirabilis atpF with interaction partners from both photosynthetic and non-photosynthetic organisms, researchers can determine whether the loss of photosynthesis has affected the molecular recognition properties of this protein .

• Complex stability: In vitro reconstitution experiments using recombinant subunits can reveal whether ATP synthase from non-photosynthetic plastids exhibits different stability properties compared to photosynthetic counterparts .

• Assembly requirements: The minimal subunit composition required for a functional ATP synthase complex in non-photosynthetic plastids may differ from that in photosynthetic plastids. Recombinant proteins allow systematic testing of different subunit combinations .

• Regulatory mechanisms: Comparative studies may reveal differences in how ATP synthase assembly and activity are regulated in photosynthetic versus non-photosynthetic contexts .

These insights contribute to our broader understanding of organelle evolution and the maintenance of essential cellular functions following major metabolic transitions.

How can genetic engineering of recombinant atpF advance research on non-photosynthetic plastids?

The ability to produce recombinant A. mirabilis atpF enables several advanced genetic engineering approaches:

• Site-directed mutagenesis: Targeted mutations can be introduced to test hypotheses about structure-function relationships, particularly regarding adaptations specific to non-photosynthetic contexts .

• Domain swapping: Chimeric proteins containing domains from photosynthetic and non-photosynthetic homologs can help identify regions responsible for specific functional properties .

• Fluorescent tagging: Fusion proteins incorporating fluorescent domains enable visualization of ATP synthase distribution and dynamics in heterologous systems .

• Affinity engineering: Modified versions of atpF with enhanced binding properties can be developed as research tools for isolating intact ATP synthase complexes from native sources .

How might comparative analysis of atpF from diverse non-photosynthetic plants inform our understanding of plastid evolution?

Aneura mirabilis represents just one evolutionary scenario of the transition to a non-photosynthetic lifestyle. Comparative analysis of atpF from diverse non-photosynthetic plants can reveal:

• Convergent adaptations: Whether similar modifications to atpF structure and function have evolved independently in different lineages following the loss of photosynthesis .

• Evolutionary rate variation: Whether the evolutionary rate of atpF changes predictably following the loss of photosynthesis, potentially indicating shifts in selective pressure .

• Correlation with ecological strategies: Whether different types of non-photosynthetic lifestyles (parasitic, mycoheterotrophic) are associated with different patterns of ATP synthase evolution .

This comparative approach requires both phylogenetic analysis and functional characterization of recombinant proteins from multiple species, representing a powerful strategy for understanding the evolution of energy metabolism in plants.

What techniques can be used to study the interaction between recombinant atpF and other ATP synthase components?

Advanced techniques for studying protein-protein interactions are particularly valuable for understanding how atpF functions within the ATP synthase complex:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This technique can map interaction surfaces between atpF and other subunits by identifying regions that show altered solvent accessibility upon binding .

• Cross-linking mass spectrometry (XL-MS): Chemical or photoreactive cross-linkers can capture transient interactions between atpF and other subunits, providing spatial constraints for structural modeling .

• Single-molecule FRET: By labeling specific sites on atpF and interaction partners with appropriate fluorophores, researchers can monitor dynamic interactions in real-time .

• Cryo-electron microscopy: Advances in cryo-EM technology now enable structural determination of membrane protein complexes at near-atomic resolution, providing insights into the organization of ATP synthase components .

These complementary approaches can provide a comprehensive picture of how atpF contributes to ATP synthase structure and function in the unique context of non-photosynthetic plastids.