Recombinant Manihot esculenta ATP synthase subunit b, chloroplastic (atpF)

What is the basic structure and function of ATP synthase subunit b in cassava chloroplasts?

ATP synthase subunit b (atpF) in Manihot esculenta chloroplasts functions as a critical component of the F0 sector of ATP synthase, anchoring the complex within the thylakoid membrane and participating in proton translocation. The protein comprises 184 amino acids with a molecular structure that includes membrane-spanning domains and interaction sites with other ATP synthase subunits. The full amino acid sequence is: MKNITDSFVSLGHWPSAGSFGFNTDILATNLINLSVVLGVLIFFGKGVLSDLLDNRKQRILDTIRNSEKLREGAIEQLEKARARLRKVEIEADQFRTNGYSEIEREKLNLINSTYKTLEQLENYKNETIHFEQQRTINQVRQRVFQQALQGALGTLNSCLTNELHLRTINANLGMFGAIKEITD . Functionally, atpF contributes to maintaining the structural integrity of the ATP synthase complex and facilitating the energy conversion process essential for photosynthesis.

How does atpF differ from other ATP synthase components, and what makes it worthy of specific investigation?

AtpF deserves specific investigation as it represents one of the crucial membrane-embedded components of the ATP synthase complex with unique structural characteristics. Unlike the catalytic subunits (α, β) that have received extensive attention, membrane subunits like atpF remain less thoroughly characterized despite their essential roles in complex assembly and function. The protein exhibits significant conservation patterns that make it valuable for evolutionary studies, similar to what has been observed with atp1/atpA genes where evidence of chloroplast-to-mitochondrial gene conversion has been documented . Additionally, studying atpF provides insights into chloroplast-specific adaptations of the ATP synthase complex that differ from mitochondrial and bacterial counterparts.

What phylogenetic information can be derived from atpF sequence analysis across plant species?

Phylogenetic analysis of atpF sequences can reveal important evolutionary relationships among plant species, particularly within the Euphorbiaceae family to which Manihot esculenta belongs. Similar to studies conducted with atp1/atpA genes, atpF analysis can potentially identify regions of high sequence conservation that might be subject to selective pressure . When constructing phylogenetic trees using atpF sequences, researchers should be aware of potential homologous recombination events that might complicate evolutionary interpretations. As demonstrated with atp1/atpA, such recombination can create functional genes of chimeric origin, potentially affecting phylogenetic reconstructions . For meaningful phylogenetic inference, atpF sequences should be analyzed alongside other chloroplast genes to establish more robust evolutionary relationships among plant species.

What expression systems yield optimal results for recombinant atpF production?

The E. coli expression system has proven effective for recombinant Manihot esculenta atpF production, as demonstrated in available protocols . For optimal expression, the following methodological approach is recommended:

• Gene optimization: Codon optimization for E. coli expression to enhance protein yield

• Vector selection: pET-series vectors with N-terminal His-tag for simplified purification

• Host strain selection: BL21(DE3) or Rosetta strains to accommodate potential rare codons

• Induction conditions: IPTG concentration of 0.5-1.0 mM at reduced temperatures (16-25°C) to minimize inclusion body formation

• Growth media: Enriched media such as Terrific Broth with appropriate antibiotics

Alternative expression systems including insect cells (baculovirus) may be considered for cases where proper folding is challenging in prokaryotic systems, though this typically comes with reduced yield compared to bacterial expression.

What purification strategies ensure high purity and biological activity of recombinant atpF?

A robust purification protocol for His-tagged recombinant atpF should include:

• Initial clarification: Sonication or high-pressure homogenization in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10% glycerol, and protease inhibitors

• Affinity chromatography: Ni-NTA purification with imidazole gradient elution (20-250 mM)

• Size exclusion chromatography: To remove aggregates and obtain homogeneous protein

• Buffer exchange: Final buffer composition optimized for stability (typically Tris/PBS-based buffer with 6% trehalose at pH 8.0)

• Quality control: SDS-PAGE analysis to confirm >90% purity

Purified protein should be stored with 5-50% glycerol (with 50% being standard) and aliquoted for long-term storage at -20°C/-80°C to prevent repeated freeze-thaw cycles that can compromise activity . Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL for experimental use .

How can researchers troubleshoot common challenges in atpF expression and purification?

When encountering issues with recombinant atpF expression and purification, consider implementing the following troubleshooting strategies:

Addressing these challenges methodically while documenting results is essential for establishing reproducible protocols for consistent atpF production.

What experimental approaches can determine the functional integrity of recombinant atpF?

Assessing the functional integrity of recombinant atpF requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Limited proteolysis to probe folding quality

  • Thermal shift assays to determine stability

• Protein-protein interaction analysis:

  • Pull-down assays with other ATP synthase components

  • Surface plasmon resonance (SPR) to quantify binding kinetics

  • Yeast two-hybrid or split-GFP assays for in vivo interaction studies

• Functional reconstitution:

  • Liposome reconstitution with purified ATP synthase components

  • ATP synthesis activity measurements in reconstituted systems

  • Proton translocation assays in proteoliposomes

These methodologies provide complementary information about different aspects of atpF functionality, with structural analysis serving as a prerequisite for more complex functional studies.

How can recombinant atpF contribute to understanding ATP synthase assembly in chloroplasts?

Recombinant atpF provides valuable tools for investigating ATP synthase assembly through:

• Assembly intermediate characterization: Using tagged recombinant atpF to identify and isolate assembly intermediates from chloroplasts, allowing mapping of the assembly pathway.

• Interaction partner identification: Employing immunoprecipitation with anti-His antibodies following incorporation of recombinant atpF into chloroplast membranes to identify novel interaction partners.

• Structure-function relationship studies: Creating site-directed mutants of recombinant atpF to identify critical residues for assembly and function through complementation studies in model systems.

• In vitro assembly systems: Developing reconstitution protocols combining recombinant atpF with other purified ATP synthase components to recreate assembly steps under controlled conditions.

These approaches collectively address the fundamental question of how the ATP synthase complex is assembled in chloroplasts and how this process is regulated during chloroplast biogenesis.

What insights can atpF research provide regarding interorganellar genetic exchange in plants?

Research on atpF can contribute significantly to understanding interorganellar genetic exchange, similar to findings observed with atp1/atpA genes. While no direct evidence of chloroplast-to-mitochondrial gene conversion has been reported for atpF specifically, the patterns observed with atp1/atpA suggest such phenomena might exist more broadly across organellar genomes. Studies on atp1/atpA revealed recurrent conversion of short patches of mitochondrial genes by chloroplast homologs during angiosperm evolution, particularly in regions of highest sequence conservation .

When investigating potential gene conversion events involving atpF, researchers should:

• Apply computational approaches similar to those developed for atp1/atpA to detect homologous recombination events involving atpF across large numbers of plant species

• Focus analysis on regions with highest nucleotide and amino acid conservation, as these are most likely to facilitate recombination and conversion events

• Consider the implications of such findings for phylogenetic analyses that use chloroplast genes, as chimeric genes resulting from interorganellar recombination can confound evolutionary interpretations

This research direction connects atpF studies to broader questions about genome evolution and the complex genetic relationships between organelles in plant cells.

What methodological approaches are most effective for studying selection pressure on atpF?

To effectively study selection pressure on atpF, researchers should implement a multi-faceted approach combining:

• Sequence-based selection tests:

  • Site-specific methods (PAML, FEL, SLAC) to identify positively selected codons

  • Branch-site models to detect lineage-specific selection

  • McDonald-Kreitman test to compare polymorphism and divergence

• Population genomics approaches:

  • FST outlier tests to identify differentiated genomic regions

  • Extended haplotype homozygosity (EHH) and integrated haplotype score (iHS) tests

  • Composite likelihood approaches like FLK and hapFLK that account for population structure

• Functional validation:

  • Site-directed mutagenesis of candidate sites under selection

  • Biochemical characterization of variant proteins

  • Physiological assessment in transgenic systems

This comprehensive approach allows for robust identification of selection signatures and subsequent functional validation, providing insights into the adaptive significance of atpF variation in cassava populations.

How does atpF evolution compare to other chloroplast genes involved in ATP synthesis?

Comparative evolutionary analysis of atpF and other chloroplast ATP synthase genes (atpA, atpB, atpE, atpH, atpI) reveals distinctive patterns of conservation and divergence:

ATP synthase genes generally show stronger conservation in catalytic domains (F1 sector) compared to membrane-embedded components (F0 sector). The atpF gene typically exhibits intermediate conservation levels, with transmembrane domains under stronger purifying selection than peripheral regions. Unlike atp1/atpA, which has documented chloroplast-to-mitochondrial gene conversion events , atpF has not been extensively studied for such interorganellar genetic exchange, representing an area for future research.

How should researchers design experiments to study atpF protein-protein interactions within the ATP synthase complex?

Designing robust experiments to investigate atpF protein-protein interactions requires multiple complementary approaches:

• In vitro interaction studies:

  • Co-immunoprecipitation with recombinant His-tagged atpF and other ATP synthase components

  • Surface plasmon resonance (SPR) with immobilized atpF to determine binding kinetics

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters of interactions

  • Crosslinking mass spectrometry (XL-MS) to identify interaction interfaces

• In vivo interaction mapping:

  • Bimolecular fluorescence complementation (BiFC) in plant protoplasts

  • Förster resonance energy transfer (FRET) with fluorescently tagged components

  • Proximity-dependent biotin identification (BioID) to capture transient interactions

• Structural studies:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction surfaces

  • Cryo-electron microscopy of reconstituted complexes with tagged atpF

  • Computational docking validated by mutagenesis of predicted interface residues

These methodologies should be implemented systematically, with initial in vitro studies validating specific interactions that can then be examined in more complex cellular contexts.

What considerations are important when designing site-directed mutagenesis studies of atpF?

When designing site-directed mutagenesis studies for atpF, researchers should consider:

• Target selection strategy:

  • Prioritize highly conserved residues identified through multi-species alignments

  • Focus on transmembrane regions and known functional domains

  • Include residues at predicted protein-protein interfaces

  • Consider sequences implicated in potential organellar gene conversion events

• Mutation design principles:

  • Conservative substitutions (e.g., L→I) to assess strict structural requirements

  • Non-conservative substitutions (e.g., D→A) to probe functional roles

  • Introduction of cysteine residues for subsequent crosslinking studies

  • Creation of phosphomimetic mutations (S/T→D/E) to investigate regulation

• Experimental validation pipeline:

  • Expression and purification assessment to evaluate structural integrity

  • Thermal stability analysis compared to wild-type protein

  • Interaction studies with known binding partners

  • Functional complementation in appropriate model systems

• Controls and statistical considerations:

  • Include multiple technical and biological replicates

  • Implement appropriate wild-type controls

  • Consider neutral mutations as negative controls

  • Perform statistical analysis appropriate for the specific assay

A well-designed mutagenesis study provides powerful insights into structure-function relationships of atpF and its role in ATP synthase assembly and function.

How can researchers effectively compare recombinant atpF properties across different plant species?

To effectively compare atpF properties across plant species, researchers should implement a standardized workflow:

• Homogeneous expression and purification:

  • Use identical expression systems and vectors for all species variants

  • Implement uniform purification protocols to minimize methodology-based variations

  • Verify comparable purity levels (>90%) by SDS-PAGE for all preparations

  • Quantify protein concentrations using consistent methods

• Comparative biophysical characterization:

  • Thermal stability assessment via differential scanning fluorimetry (DSF)

  • Secondary structure analysis by circular dichroism (CD) spectroscopy

  • Hydrodynamic properties determination through size exclusion chromatography

  • Membrane interaction studies using liposome binding assays

• Functional comparison:

  • Standardized binding assays with conserved interaction partners

  • ATP synthase reconstitution with homologous or heterologous components

  • Activity measurements under identical experimental conditions

• Data analysis and presentation:

  • Statistical comparison across species using appropriate tests

  • Normalization strategies to account for experimental variation

  • Correlation of functional differences with sequence divergence

  • Phylogenetic context integration for evolutionary interpretation

This systematic approach ensures that observed differences reflect genuine species-specific adaptations rather than methodological artifacts.

How can atpF research contribute to understanding cassava adaptation to environmental stress?

AtpF research offers important insights into cassava's environmental adaptation mechanisms through several research avenues:

• Stress-responsive expression analysis:

  • Quantification of atpF transcript and protein levels under various stress conditions

  • Correlation of expression patterns with photosynthetic efficiency

  • Comparison between stress-tolerant and susceptible cassava varieties

• Genetic diversity studies:

  • Analysis of atpF sequence variation across cassava varieties from diverse environments

  • Identification of SNPs associated with performance under stress conditions

  • Integration with broader genomic diversity studies of cassava populations

• Physiological investigations:

  • Assessment of ATP synthase function in stress-exposed plants

  • Correlation of enzyme activity with growth performance under stress

  • Analysis of energy balance maintenance during environmental challenges

• Biotechnological applications:

  • Development of atpF-based markers for stress tolerance breeding

  • Exploration of genetic engineering approaches targeting ATP synthase components

  • Design of screening methods for identifying resilient cassava varieties

This research direction connects molecular studies of atpF to the practical challenges of improving cassava cultivation in changing environmental conditions, particularly in traditional growing regions where cassava serves as a staple crop .

What emerging technologies could advance atpF structural and functional research?

Several cutting-edge technologies promise to significantly advance atpF research:

• Cryo-electron microscopy advancements:

  • Single-particle analysis reaching near-atomic resolution

  • Tomography approaches for in situ visualization of ATP synthase in thylakoid membranes

  • Time-resolved structures capturing different functional states

• Integrative structural biology:

  • Combining X-ray crystallography, NMR, and computational modeling

  • Hydrogen-deuterium exchange mass spectrometry for dynamic structural information

  • Integrative modeling platforms incorporating diverse experimental constraints

• Advanced genetic technologies:

  • CRISPR-Cas9 precise genome editing of plastid genomes

  • Base editing for introducing specific atpF mutations without double-strand breaks

  • Optogenetic control of ATP synthase components

• Single-molecule techniques:

  • High-speed atomic force microscopy for real-time visualization

  • Magnetic tweezers for measuring mechanical properties of individual complexes

  • Single-molecule FRET for conformational dynamics studies

These technologies collectively address current limitations in understanding atpF structure, dynamics, and function within the ATP synthase complex and plant cellular context.

How might research on atpF inform synthetic biology approaches to chloroplast engineering?

Research on atpF provides valuable insights for synthetic biology approaches to chloroplast engineering through:

• Minimal ATP synthase design:

  • Identification of essential components and interactions required for function

  • Development of simplified ATP synthase complexes with reduced subunit composition

  • Creation of hybrid complexes incorporating engineered atpF variants

• Energy efficiency optimization:

  • Engineering modified atpF proteins with altered proton/ATP ratios

  • Designing synthetic regulatory circuits controlling ATP synthase assembly

  • Creating environment-responsive ATP production systems

• Chloroplast transformation platforms:

  • Development of selection markers based on atpF function

  • Design of synthetic operons incorporating optimized atpF genes

  • Creation of orthogonal expression systems for chloroplast engineering

• Bioenergetic circuit design:

  • Integration of engineered ATP synthase components with other photosynthetic complexes

  • Development of synthetic metabolic pathways powered by optimized ATP production

  • Creation of artificial regulatory systems controlling energy production

These applications represent the translation of fundamental atpF research into biotechnological innovations that could enhance crop productivity, create bio-based energy solutions, and develop new platforms for sustainable bioproduction.

What are the major challenges in expressing and purifying functional recombinant atpF, and how can they be addressed?

Expressing and purifying functional recombinant atpF presents several technical challenges with specific solutions:

Addressing these challenges requires systematic optimization and well-designed quality control measures to ensure that the recombinant protein accurately represents the native atpF in structural and functional properties.

How can researchers effectively study the integration of atpF into the ATP synthase complex?

Studying atpF integration into the ATP synthase complex requires specialized approaches:

• In vitro reconstitution systems:

  • Sequential addition of purified components to monitor assembly steps

  • Fluorescently labeled atpF to track incorporation into complexes

  • Affinity-tagged atpF for pull-down of assembly intermediates

  • Detergent-based reconstitution followed by proteoliposome formation

• Cellular import and assembly assays:

  • Isolated chloroplast import experiments with radiolabeled or fluorescent atpF

  • Time-course analysis of incorporation into complexes following import

  • Competition assays with mutant variants to identify critical assembly determinants

  • Pulse-chase experiments to determine assembly kinetics

• Advanced microscopy techniques:

  • Super-resolution imaging of tagged ATP synthase components

  • Single-particle tracking of labeled atpF during assembly

  • FRET-based biosensors reporting on successful complex formation

  • Correlative light and electron microscopy for ultrastructural context

• Quantitative analysis methods:

  • Mathematical modeling of assembly pathways

  • Kinetic analysis of complex formation under various conditions

  • Statistical evaluation of assembly efficiency for different variants

  • Machine learning approaches to identify assembly patterns

These methodologies collectively provide a comprehensive view of how atpF becomes incorporated into functional ATP synthase complexes within chloroplasts.

What quality control measures ensure reliable results in atpF research?

Implementing rigorous quality control measures is essential for obtaining reliable results in atpF research:

• Protein quality assessment:

  • SDS-PAGE to verify >90% purity and expected molecular weight

  • Mass spectrometry to confirm protein identity and detect modifications

  • Size exclusion chromatography to assess oligomeric state and aggregation

  • Circular dichroism to verify proper secondary structure content

• Functional validation:

  • ATP synthesis activity measurements with reconstituted complexes

  • Binding assays with known interaction partners

  • Conformational change dynamics using appropriate biophysical techniques

  • Comparison with native ATP synthase complexes from chloroplasts

• Experimental design considerations:

  • Inclusion of appropriate positive and negative controls

  • Multiple biological and technical replicates

  • Blinding procedures for subjective assessments

  • Randomization of sample processing order

• Data analysis and reporting:

  • Statistical analysis appropriate for the experimental design

  • Transparent reporting of all experimental conditions

  • Sharing of detailed protocols for reproducibility

  • Availability of raw data and analysis scripts

Adherence to these quality control measures ensures that research findings on atpF are robust, reproducible, and make meaningful contributions to our understanding of ATP synthase biology in Manihot esculenta and other plant species.