Recombinant Cyanidioschyzon merolae ATP synthase subunit b', chloroplastic (atpG)

What is the role of ATP synthase subunit b' (atpG) in Cyanidioschyzon merolae chloroplasts?

ATP synthase subunit b' (atpG) in C. merolae is a critical component of the peripheral stalk of the chloroplastic ATP synthase complex. This subunit provides essential structural support that maintains the integrity between the F₁ catalytic domain and the F₀ membrane-embedded proton channel. Studies have shown that the peripheral stalk, which includes subunit b', is crucial for stabilizing the c-ring/F₁ complex during the rotary catalytic mechanism that drives ATP synthesis .

Research indicates that in the absence of functional peripheral stalk subunits, ATP synthase cannot properly assemble or function. The gene encoding this subunit in C. merolae is nuclear, unlike some other ATP synthase components that are encoded in the chloroplast genome, representing an interesting example of coordinated expression between nuclear and organellar genomes .

How is C. merolae atpG expression regulated under different environmental conditions?

C. merolae is an extremophilic red alga capable of surviving at high temperatures (40°C) and low pH (2-3) . The expression of atpG responds to these extreme environmental conditions through several regulatory mechanisms:

The regulatory elements in the atpG promoter region likely contain binding sites for transcription factors responsive to stress conditions. Analysis of the 5' UTR of atpG transcripts reveals potential sites for alternative polyadenylation, which may contribute to transcript stability under stress conditions .

What are the most effective methods for cloning and expressing recombinant C. merolae atpG?

The following protocol has been optimized for successful cloning and expression of recombinant C. merolae atpG:

• Gene Amplification:

  • Extract genomic DNA from C. merolae using modified acid phenol method

  • Design primers that flank the atpG coding sequence with appropriate restriction sites

  • Use high-fidelity DNA polymerase for PCR amplification (initial denaturation: 98°C for 2 min; 30 cycles of 98°C for 10 sec, 60°C for 30 sec, 72°C for 1 min; final extension: 72°C for 5 min)

• Expression Vector System:

  • The pCAT vector system has been optimized for C. merolae transformation, utilizing chloramphenicol acetyltransferase as a selection marker

  • For heterologous expression, the pET-28a vector with an N-terminal His-tag has shown good results

• Transformation Protocol:

  • For homologous recombination in C. merolae, use the optimized CAT transformation protocol that yields chloramphenicol-resistant transformants in under two weeks

  • Target the convergent intergenic region of CMD184C and CMD185C for stable expression

  • Utilize the authentic Cm-Cm URA5.3 gene (URA) as a selection marker for single-copy insertion rather than the chimeric URA marker (Cm-Gs URA) which can cause multicopy insertion

What purification strategies yield the highest quality recombinant atpG protein?

For optimal purification of recombinant C. merolae atpG:

• Cell Lysis:

  • For C. merolae cells: Sonication in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 10% glycerol, 1 mM PMSF, and protease inhibitor cocktail

  • For E. coli expression systems: BugBuster reagent with lysozyme has shown good results

• Chromatography Steps:

  • Initial purification: Ni-NTA affinity chromatography for His-tagged constructs

  • Secondary purification: Ion exchange chromatography (Q-Sepharose)

  • Final polishing: Size exclusion chromatography using Superdex 200

• Quality Assessment:

  • SDS-PAGE analysis: Expected molecular weight for atpG is approximately 20 kDa

  • Western blot using antibodies against conserved ATP synthase subunit b' epitopes

  • Circular dichroism spectroscopy to confirm proper folding

How can CRISPR-Cas9 gene editing be optimized for studying atpG function in C. merolae?

CRISPR-Cas9 gene editing in C. merolae requires specialized approaches:

• Design Considerations:

  • Select guide RNAs with high specificity scores (>85) targeting the atpG coding region

  • Avoid potential off-target sites by checking against the complete C. merolae genome

  • Design repair templates with at least 500 bp homology arms on either side of the cut site

• Delivery Methods:

  • Polyethylene glycol (PEG)-mediated transformation has proven effective for C. merolae

  • Use the Cm-Cm URA5.3 selection system for single-copy integration events

  • Maintain selection pressure throughout the culture process

• Screening Strategy:

  • Initial screening by colony PCR using primers flanking the target site

  • Confirmation by Southern blotting to verify single-copy integration

  • Sequence verification of the entire modified locus

CRISPR-Cas9 knockout of atpG would likely be lethal, as seen in related ATP synthase subunit studies where disruption of peripheral stalk subunits fully prevents ATP synthase function and accumulation . Therefore, conditional knockdown approaches may be more informative.

What protein-protein interaction techniques are most suitable for investigating atpG associations within the ATP synthase complex?

Several techniques have been optimized for studying atpG interactions:

• Co-immunoprecipitation (Co-IP):

  • Generate antibodies against C. merolae atpG or use epitope-tagged versions

  • Extract protein complexes under mild detergent conditions (0.5% digitonin or 1% n-dodecyl β-D-maltoside)

  • Identify interacting partners by mass spectrometry

• Crosslinking Mass Spectrometry:

  • Use membrane-permeable crosslinkers (DSS or BS3) at optimized concentrations

  • Digest crosslinked complexes with trypsin and enrich crosslinked peptides

  • Analyze by LC-MS/MS to identify distance constraints between subunits

• Bimolecular Fluorescence Complementation (BiFC):

  • Split fluorescent protein fusions to atpG and potential interaction partners

  • Express in C. merolae using the optimized transformation protocol

  • Visualize interactions using fluorescence microscopy

• Cryo-EM Analysis:

  • Purify intact ATP synthase complexes using digitonin solubilization

  • Apply specimens to holey carbon grids and vitrify in liquid ethane

  • Collect and process images to generate 3D reconstructions of the complex

These methods have revealed that atpG interacts primarily with other peripheral stalk components and provides critical contacts with both the membrane-embedded F₀ sector and the catalytic F₁ sector .

How does the function of atpG differ between C. merolae and other photosynthetic organisms?

Comparative analysis reveals several unique features of C. merolae atpG:

C. merolae atpG contains unique residues that likely contribute to its stability under extreme conditions. The protein maintains proper folding and function at temperatures up to 40°C and pH as low as 2, conditions that would denature ATP synthase components from most other organisms .

Unlike some other photosynthetic organisms, C. merolae has a highly reduced genome with minimal intergenic spaces and no introns in its plastid genes . The nuclear-encoded atpG gene in C. merolae is part of a tightly regulated expression system that coordinates with plastid-encoded ATP synthase components through mechanisms that may involve alternative polyadenylation .

How can researchers address the challenges of expressing thermostable and acid-stable atpG for structural studies?

The expression of functional C. merolae atpG for structural studies presents unique challenges:

• Stability Optimization:

  • Incorporate native C. merolae chaperones (e.g., Hsp60 family proteins) during expression

  • Use acidic buffer systems (pH 4.0-5.0) during purification to maintain native conformation

  • Add stabilizing agents such as glycerol (10-20%) and specific lipids during purification

• Co-expression Strategies:

  • Co-express atpG with interacting subunits to form stable sub-complexes

  • Utilize dual-vector systems encoding multiple ATP synthase components

  • Engineer fusion constructs that preserve critical interaction domains

• Crystallization Approaches:

  • Screen detergent conditions extensively (focus on maltoside and glucoside detergents)

  • Implement lipidic cubic phase crystallization for membrane-associated regions

  • Apply microseed matrix screening to optimize crystal growth conditions

What approaches can resolve contradictory data regarding the topology of atpG within the ATP synthase complex?

Researchers have reported conflicting models of atpG orientation within the ATP synthase complex. To resolve these contradictions:

• Integrated Structural Analysis:

  • Combine cryo-EM of the intact complex with X-ray crystallography of individual components

  • Apply cross-linking mass spectrometry to establish distance constraints

  • Use hydrogen-deuterium exchange mass spectrometry to map exposed surfaces

• In vivo Probing:

  • Implement site-specific fluorescent labeling at putative membrane-proximal regions

  • Apply accessibility studies using membrane-impermeable modifying reagents

  • Create systematic deletion constructs to identify essential structural elements

• Computational Approaches:

  • Develop molecular dynamics simulations incorporating the unique lipid composition of C. merolae

  • Apply evolutionary coupling analysis to identify co-evolving residue pairs

  • Generate integrated models that satisfy all experimental constraints

Current evidence suggests that C. merolae atpG adopts an extended α-helical structure that spans from the membrane surface to the F₁ catalytic domain, consistent with its role in preventing rotation of the α₃β₃ hexamer during ATP synthesis .

How can the unique properties of C. merolae atpG inform the engineering of ATP synthase for biotechnological applications?

The exceptional stability of C. merolae ATP synthase components presents opportunities for biotechnological applications:

• Stability Transfer:

  • Identify specific residues in C. merolae atpG responsible for thermostability

  • Transfer these features to ATP synthase components from other organisms

  • Test chimeric constructs for enhanced stability while maintaining function

• Nanomotor Applications:

  • Exploit the robust nature of C. merolae ATP synthase for nanomotor development

  • Engineer modified versions with controlled rotation rates under extreme conditions

  • Incorporate synthetic interfaces for coupling to non-biological components

• Bioenergetic Systems:

  • Develop artificial photosynthetic systems incorporating C. merolae ATP synthase

  • Create hybrid energy-harvesting devices that function under acidic conditions

  • Explore integration with industrial processes that operate at elevated temperatures

The exceptional ability of C. merolae ATP synthase to function under conditions where "most other organisms would die" makes it an attractive platform for developing robust bioenergetic systems for extreme environments.

What are the optimal gene targeting strategies for atpG modification in C. merolae?

Based on comprehensive studies of C. merolae transformation:

• Homologous Recombination Approach:

  • Use the authentic Cm-Cm URA5.3 gene (URA) as a selection marker rather than the chimeric URA marker (Cm-Gs URA)

  • This approach has been demonstrated to yield single-copy insertion at targeted loci with high reliability

  • Design homology arms of at least 500 bp on either side of the insertion site

• Target Selection:

  • The convergent intergenic region of CMD184C and CMD185C has been validated as an effective target site for stable expression

  • When targeting atpG directly, consider using conditional approaches as complete knockout may be lethal

• Verification of Transformants:

  • Perform colony PCR using primers that span the insertion junctions

  • Conduct Southern blotting to confirm single-copy integration

  • Sequence the entire modified locus to verify the absence of unintended mutations

How can researchers quantitatively assess atpG expression levels and protein abundance?

Several complementary approaches have been optimized for C. merolae:

• Transcript Analysis:

  • qRT-PCR using primers specific to atpG (efficiency = 98.2±1.3%)

  • RNA-Seq analysis with appropriate normalization to stable reference genes

  • Northern blotting to detect potential alternative transcripts, as C. merolae shows evidence of alternative polyadenylation

• Protein Quantification:

  • Western blotting with anti-atpG antibodies, normalized to total protein

  • Mass spectrometry-based approaches using labeled reference peptides

  • Green fluorescent protein fusions for in vivo visualization and semi-quantitative analysis

• Functional Assessment:

  • ATP synthesis rate measurements in isolated chloroplasts

  • Membrane potential measurements using fluorescent dyes

  • Oxygen evolution as a proxy for photosynthetic ATP production

Expected atpG protein abundance in wildtype C. merolae is approximately 2-5% of total chloroplast membrane protein, with expression levels varying based on growth conditions and developmental stage.

How can researchers reconcile contradictory findings regarding atpG stoichiometry in the ATP synthase complex?

Conflicting reports exist regarding the exact stoichiometry of atpG in C. merolae ATP synthase. To address these contradictions:

• Quantitative Analysis Approaches:

  • Apply absolute quantification using mass spectrometry with isotope-labeled standards

  • Perform single-molecule photobleaching of fluorescently tagged subunits

  • Use genetic titration experiments with controlled expression levels

• Structural Validation:

  • Implement high-resolution cryo-EM studies of the intact complex

  • Apply mass photometry to determine subunit stoichiometry in native complexes

  • Use chemical crosslinking followed by SDS-PAGE to identify subunit interactions

• Functional Correlation:

  • Measure ATP synthesis rates with varying levels of atpG expression

  • Assess proton conductance as a function of atpG content

  • Evaluate complex stability under stress conditions with different atpG stoichiometries

The most current evidence suggests that C. merolae ATP synthase contains two copies of atpG per complex, consistent with the general architecture of F-type ATP synthases, though definitive structural data specific to C. merolae is still being developed.

What experimental designs can resolve conflicts in the literature regarding atpG post-translational modifications?

Several approaches can help clarify the contradictory reports about atpG post-translational modifications:

• Comprehensive PTM Mapping:

  • Employ multiple proteases for complete sequence coverage

  • Use complementary mass spectrometry fragmentation techniques (CID, ETD, HCD)

  • Apply targeted MS/MS approaches to focus on regions of interest

• Modification-Specific Analyses:

  • Develop antibodies specific to putative modification sites

  • Generate site-directed mutants of predicted modification sites

  • Use chemical approaches to selectively enrich modified peptides

• Functional Correlation Studies:

  • Compare PTM profiles under different physiological conditions

  • Assess the impact of mutations at modification sites on ATP synthase function

  • Monitor changes in PTM patterns during stress responses

Current evidence suggests that phosphorylation of C. merolae atpG may occur at conserved threonine residues in the C-terminal domain, potentially regulating interactions with other peripheral stalk components, though this requires further experimental validation.

How can emerging technologies enhance our understanding of atpG function in C. merolae?

Recent technological advances offer new opportunities for atpG research:

• Cryo-Electron Tomography:

  • Apply focused ion beam milling to prepare C. merolae cells

  • Visualize ATP synthase in its native cellular context

  • Determine the spatial organization of ATP synthase complexes in the thylakoid membrane

• Single-Molecule Approaches:

  • Implement high-speed AFM to observe conformational dynamics

  • Apply magnetic tweezers to measure mechanical properties of individual complexes

  • Use FRET pairs to monitor subunit movements during catalysis

• Advanced Genetic Tools:

  • Develop inducible gene expression systems for C. merolae

  • Apply optogenetic approaches to control ATP synthase assembly

  • Implement CRISPR interference for targeted gene repression

What bioinformatic approaches provide insights into atpG evolution and adaptation to extreme environments?

Computational methods reveal evolutionary insights about C. merolae atpG:

• Comparative Genomics:

  • Phylogenetic analysis of atpG sequences across diverse algal lineages

  • Identification of signature residues associated with thermostability

  • Analysis of selection pressure on different domains of the protein

• Structural Bioinformatics:

  • Homology modeling based on available ATP synthase structures

  • Molecular dynamics simulations under varying temperature and pH conditions

  • Prediction of stabilizing interactions unique to C. merolae atpG

• Systems Biology Integration:

  • Correlation of atpG expression with global transcriptomic responses to stress

  • Network analysis of co-expressed genes under extreme conditions

  • Metabolic modeling to predict the impact of atpG variants on cellular energetics

These analyses suggest that C. merolae atpG has undergone adaptive evolution specifically in regions that interact with the membrane domain, likely contributing to its remarkable stability under extreme conditions.