Recombinant Nicotiana tomentosiformis ATP synthase subunit b, chloroplastic (atpF)

What is the evolutionary significance of studying ATP synthase subunit b in Nicotiana tomentosiformis?

N. tomentosiformis serves as one of the ancestral species of commercial tobacco (Nicotiana tabacum), which formed through interspecific hybridization approximately 200,000 years ago. Studying the chloroplastic ATP synthase subunit b in this species provides critical evolutionary insights into energy metabolism across Nicotiana species . N. tomentosiformis has been primarily investigated as a progenitor of N. tabacum, and its chloroplast proteins represent an important model for understanding the evolution of bioenergetic systems in the Solanaceae family .

The atpF gene encoding ATP synthase subunit b is particularly valuable for evolutionary studies because it participates in the essential process of ATP production via the electrochemical proton gradient generated by photosynthesis. Comparative analyses of atpF between N. tomentosiformis and other Nicotiana species can illuminate patterns of selection pressure on energy-producing systems and the adaptive significance of specific protein variants in different environmental contexts.

How does N. tomentosiformis atpF differ structurally and functionally from other Nicotiana species?

The ATP synthase subunit b from N. tomentosiformis exhibits structural distinctions that reflect its specialized role in the chloroplastic ATP synthase complex. While maintaining the core functional domains required for ATP synthesis, the protein contains sequence variations that may contribute to species-specific regulation and efficiency of energy production.

In chloroplast ATP synthase, subunit b forms part of the peripheral stalk that connects the membrane-embedded Fo motor to the catalytic F1 head . This structure helps redistribute differences in torsional energy across three unequal steps in the rotation cycle during ATP synthesis . When comparing sequence homology between N. tomentosiformis and N. sylvestris atpF, specific amino acid substitutions are observed, potentially reflecting adaptations to different ecological niches occupied by these species throughout their evolution .

The functional implications of these structural differences may include:

• Altered stability of the ATP synthase complex under varying environmental conditions

• Different efficiencies in proton translocation and ATP synthesis

• Varied sensitivity to regulatory mechanisms that control energy production in response to light and metabolic demands

• Species-specific interactions with other components of the photosynthetic apparatus

What are the optimal expression systems for producing recombinant N. tomentosiformis atpF protein?

The selection of an appropriate expression system for recombinant N. tomentosiformis atpF is critical for obtaining functional protein. Based on established methodologies for chloroplast proteins, several expression systems can be considered:

Table 1. Comparison of Expression Systems for Recombinant N. tomentosiformis atpF

For functional studies requiring properly folded protein with authentic post-translational modifications, plant-based expression systems provide significant advantages despite lower yields. When protein quantity is prioritized over native folding, E. coli systems with codon optimization for plant chloroplast proteins can be effective . Expression in E. coli should be performed at lower temperatures (16-18°C) to minimize inclusion body formation, using specialized vectors that incorporate chloroplast transit peptides to improve folding.

The addition of molecular chaperones as co-expression partners can significantly improve the solubility and proper folding of recombinant atpF in bacterial systems. Additionally, fusion tags such as MBP (maltose-binding protein) or SUMO often enhance solubility while allowing for subsequent tag removal via specific proteases.

What purification strategy yields the highest purity and activity of recombinant N. tomentosiformis atpF?

A multi-step purification strategy is essential for obtaining high-purity, functional recombinant atpF protein:

• Initial capture: Affinity chromatography using nickel-NTA resin for His-tagged proteins achieves efficient initial purification with binding buffer containing 20 mM Tris-HCl pH 8.0, 300 mM NaCl, 20 mM imidazole, and 5% glycerol .

• Intermediate purification: Ion exchange chromatography using a gradient of 50-500 mM NaCl separates the target protein from contaminants with similar affinity properties but different charge characteristics.

• Polishing step: Size exclusion chromatography with a Superdex 200 column equilibrated in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, and 1 mM DTT provides final purification and buffer exchange.

For membrane protein purification, it's critical to include appropriate detergents throughout the process. A comparative analysis of detergent effectiveness shows:

Table 2. Detergent Selection for atpF Purification

Activity assays should be performed after each purification step to ensure the protein maintains its functional integrity. The yield typically ranges from 1-5 mg of purified protein per liter of culture, with purity exceeding 95% as assessed by SDS-PAGE and analytical SEC.

How can researchers effectively analyze the structure-function relationship of recombinant N. tomentosiformis atpF?

Elucidating the structure-function relationship of recombinant N. tomentosiformis atpF requires a multi-faceted approach combining structural biology techniques with functional assays:

• Structural analysis:

  • Cryo-electron microscopy (Cryo-EM) offers exceptional resolution for visualizing the integration of atpF within the complete ATP synthase complex, revealing side chains of all protein subunits and the nucleotides in the F1 head .

  • X-ray crystallography of isolated atpF or subcomplexes provides atomic resolution of critical domains.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) identifies regions with different solvent accessibility, indicating structural flexibility important for function.

  • Circular dichroism (CD) spectroscopy determines secondary structure composition (α-helices, β-sheets) under various conditions.

• Functional assessment:

  • ATP synthesis assays using reconstituted liposomes measure the protein's ability to support ATP production driven by an artificially generated proton gradient.

  • ATPase activity assays assess the reverse reaction, hydrolysis of ATP, which can be measured using malachite green phosphate detection methods similar to those used for ATP sulfurylase .

  • Proton translocation assays using pH-sensitive fluorescent dyes monitor the protein's ability to facilitate proton movement across membranes.

• Structure-function correlation:

  • Site-directed mutagenesis of conserved and variable residues between N. tomentosiformis and other species identifies amino acids critical for function.

  • Domain swapping experiments with homologous proteins from other species (e.g., N. sylvestris) determine the functional significance of species-specific regions.

  • Cross-linking studies coupled with mass spectrometry map interaction interfaces with other ATP synthase subunits.

The integration of these approaches allows researchers to develop comprehensive models of how specific structural features of N. tomentosiformis atpF contribute to its function within the ATP synthase complex and how these features may differ from other Nicotiana species, potentially reflecting adaptive evolution.

What methods can effectively determine the oligomeric state and stability of recombinant N. tomentosiformis atpF?

Determining the oligomeric state and stability of recombinant atpF is crucial for understanding its assembly and function within the ATP synthase complex. Multiple complementary techniques provide robust characterization:

• Analytical ultracentrifugation (AUC) offers precise determination of molecular mass and shape in solution, distinguishing between monomeric and oligomeric forms. Sedimentation velocity experiments should be conducted at 42,000 rpm at 20°C, with scans recorded at 280 nm at 6-minute intervals.

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) provides absolute molecular weight measurement independent of shape, accurately determining oligomeric state using a Superdex 200 increase column with flow rate of 0.5 ml/min.

• Native mass spectrometry determines precise mass and stoichiometry of intact protein complexes under non-denaturing conditions. Samples should be buffer-exchanged into 200 mM ammonium acetate (pH 7.5) with 0.5% C8E4 detergent prior to analysis.

• Differential scanning fluorimetry (DSF) assesses thermal stability under various conditions:

Table 3. Effect of Environmental Factors on atpF Stability (Tm values in °C)

• Chemical cross-linking with BS3 or glutaraldehyde followed by SDS-PAGE analysis visualizes oligomeric species that exist in solution. Optimal cross-linking occurs with 0.5-2 mM BS3 for 30 minutes at room temperature.

• Blue native PAGE separates intact protein complexes under non-denaturing conditions, preserving weak interactions and revealing higher-order assemblies. Sample preparation should include 0.1% digitonin or DDM to maintain membrane protein solubility.

When interpreting these results, researchers should consider that atpF functions as part of a larger complex in vivo, and its stability and oligomeric state may differ when isolated versus in the context of the complete ATP synthase assembly. Comparisons with equivalent subunits from other species can provide valuable insights into evolutionary adaptations that affect stability and assembly.

How do ATP synthase subunit b proteins differ between N. tomentosiformis and N. sylvestris, and what are the functional implications?

Comparative analysis of ATP synthase subunit b between these two ancestral tobacco species reveals important evolutionary adaptations that influence energy metabolism:

N. tomentosiformis and N. sylvestris, as the paternal and maternal donors of N. tabacum respectively, show distinct sequence variations in their ATP synthase components that reflect their different evolutionary histories . Specific differences in the atpF protein include:

Table 4. Key Differences Between N. tomentosiformis and N. sylvestris atpF

These differences likely contribute to the distinct bioenergetic properties of these species. N. sylvestris, with its higher abundance of alkaloids (82% of 4.8 mg/g total alkaloids as nicotine), may require different energy metabolism patterns compared to N. tomentosiformis, which has lower nicotine content (6% of 0.5 mg/g total alkaloids) . The differential expression patterns of genes involved in energy metabolism between these species further support this hypothesis.

The structural variations in atpF may also contribute to adaptations to different light intensities and environmental stresses, potentially explaining some of the ecological differentiation between these species. Research investigating the photosynthetic efficiency and stress responses of recombinant ATP synthases containing either variant would provide valuable insights into these adaptive differences.

What approaches are most effective for analyzing evolutionary conservation and divergence of atpF across Nicotiana species?

Investigating evolutionary patterns of atpF across Nicotiana species requires integrating multiple computational and experimental approaches:

• Sequence-based phylogenetic analysis:

  • Multiple sequence alignment using MUSCLE or MAFFT algorithms with gap penalties optimized for transmembrane proteins

  • Maximum likelihood phylogenetic tree construction using RAxML or IQ-TREE with appropriate substitution models (LG+G+F or MTREV+G+F)

  • Calculation of synonymous (dS) and non-synonymous (dN) substitution rates to identify signatures of selection

• Structural conservation mapping:

  • Homology modeling based on the high-resolution cryo-EM structure of chloroplast ATP synthase

  • Conservation analysis using ConSurf or Evolutionary Trace methods to map conserved and variable regions onto 3D structures

  • Analysis of co-evolving residue networks using methods like GREMLIN or EVcouplings to identify functionally linked positions

• Experimental comparative biochemistry:

  • Recombinant expression of atpF variants from different Nicotiana species under identical conditions

  • Comparative enzymatic assays measuring ATP synthesis rates under standardized conditions

  • Thermal stability comparisons using differential scanning fluorimetry

  • Chimeric protein construction to identify domains responsible for species-specific functional properties

Table 5. Conservation Analysis of Key Functional Domains in Nicotiana atpF Proteins

*Conservation score scale: 1 (highly variable) to 9 (highly conserved)

This integrated approach reveals that core functional domains essential for ATP synthase activity show strong evolutionary conservation across Nicotiana species, while peripheral regions display greater variability, potentially reflecting species-specific adaptations to different ecological niches and metabolic requirements.

How can researchers effectively use recombinant N. tomentosiformis atpF to investigate chloroplast bioenergetics under various stress conditions?

Recombinant N. tomentosiformis atpF serves as a valuable tool for investigating chloroplast bioenergetic responses to environmental stresses through several methodological approaches:

• In vitro reconstitution systems:

  • Purified recombinant atpF can be incorporated into liposomes along with other ATP synthase components to create a minimal functional system

  • These reconstituted systems allow precise control over lipid composition, pH gradients, and other parameters

  • Researchers can systematically test how specific stress conditions (temperature, pH, salt, reactive oxygen species) affect ATP synthesis efficiency

  • The advantage of this approach is the ability to isolate specific components of the stress response pathway

• Ex vivo chloroplast complementation:

  • Isolated chloroplasts from model plants can be partially depleted of endogenous ATP synthase components

  • Supplementation with recombinant N. tomentosiformis atpF allows assessment of its function in a more native environment

  • This approach enables comparison of atpF performance under various stress conditions while maintaining the complex chloroplast environment

• Heterologous expression in cyanobacteria:

  • Expression of N. tomentosiformis atpF in cyanobacterial systems allows for whole-cell bioenergetic studies

  • Measurements of photosynthetic parameters (oxygen evolution, fluorescence, P700 oxidation) can be correlated with ATP synthesis under stress

  • This system bridges the gap between in vitro studies and more complex plant systems

• Transgenic studies:

  • Generation of transgenic plants expressing N. tomentosiformis atpF variants provides the most physiologically relevant system

  • Combined with knockdown/knockout of endogenous atpF, this approach allows assessment of the protein's contribution to stress tolerance

  • High-throughput phenotyping under various stress conditions can identify specific advantages or disadvantages of the N. tomentosiformis variant

Table 6. Experimental Design for Stress Response Studies with Recombinant atpF

These approaches provide complementary insights into how N. tomentosiformis atpF contributes to bioenergetic responses under stress, potentially revealing adaptations specific to this species that could be leveraged for crop improvement.

What strategies can researchers employ to investigate potential contradictions in experimental results when studying recombinant N. tomentosiformis atpF?

When researchers encounter contradictory results in studies of recombinant N. tomentosiformis atpF, systematic approaches can help resolve these discrepancies:

• Categorizing contradiction types:
  According to research on apparent contradictions in biomedical literature, contradictions can be classified into several categories :

  • Internal to the experimental system (species differences, protein isoforms)

  • External to the system (experimental conditions, reagent variations)

  • Endogenous/exogenous factors (post-translational modifications, interaction partners)

  • Known controversies in the field

  • Actual contradictions in literature requiring resolution

• Methodological standardization:

  • Develop a standardized expression and purification protocol that is consistently applied across laboratories

  • Establish reference samples of recombinant atpF with verified activity for inter-laboratory calibration

  • Create detailed standard operating procedures (SOPs) for functional assays with specific acceptance criteria

• Multivariate experimental design:

  • Implement factorial experimental designs that systematically vary multiple parameters simultaneously

  • Use statistical approaches like ANOVA to identify significant factors affecting experimental outcomes

  • Apply machine learning techniques to identify complex patterns in experimental data that might explain contradictions

• Controlled comparison studies:

  • When contradictory results arise, design head-to-head comparisons under identical conditions

  • Include positive and negative controls that establish the valid range for experimental outcomes

  • Blind experimenters to previous results to minimize confirmation bias

Table 7. Decision Matrix for Resolving Contradictory Results

• Meta-analytical approaches:

  • When sufficient data exist across multiple studies, conduct formal meta-analyses

  • Apply Bayesian methods to estimate the probability of different hypotheses explaining the contradictions

  • Identify moderator variables that systematically affect experimental outcomes across studies

By systematically applying these approaches, researchers can transform apparent contradictions into valuable insights about the factors influencing N. tomentosiformis atpF function, potentially revealing important regulatory mechanisms or structural dependencies not previously appreciated.

What are the most common technical challenges in working with recombinant N. tomentosiformis atpF and how can they be overcome?

Working with recombinant membrane proteins like N. tomentosiformis atpF presents several technical challenges that require specific troubleshooting approaches:

• Low expression yield:

  • Challenge: Hydrophobic membrane proteins often express poorly in heterologous systems

  • Solution: Optimize codon usage for the expression host; test different promoters and signal sequences; use specialized strains like C41(DE3) or C43(DE3) designed for membrane protein expression; implement auto-induction media; lower expression temperature to 16-18°C

• Protein misfolding and aggregation:

  • Challenge: Formation of inclusion bodies due to improper folding

  • Solution: Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ); use fusion partners that enhance solubility (MBP, SUMO, TrxA); implement on-column refolding during purification; add glycerol or specific lipids to stabilize native conformation

• Detergent selection challenges:

  • Challenge: Finding detergents that efficiently extract the protein while maintaining its functional state

  • Solution: Screen detergent panels using thermal shift assays; implement detergent exchange during purification; use mild detergents like LMNG or digitonin for final steps; consider amphipols or nanodiscs for enhanced stability

• Instability during purification:

  • Challenge: Loss of activity during multi-step purification

  • Solution: Minimize purification steps; maintain consistent low temperature (4°C); add stabilizing agents (glycerol, ATP, specific lipids); implement on-line activity monitoring during purification; use rapid purification approaches like tandem affinity tags

• Functional assay limitations:

  • Challenge: Difficulty in measuring activity of isolated subunit b outside of the complete ATP synthase complex

  • Solution: Develop binding assays with partner subunits; use structural probes (fluorescent labels, EPR spin labels) to monitor conformation; implement reconstitution with minimal partner proteins required for measurable function

Table 8. Troubleshooting Guide for Common Issues with Recombinant atpF

• Partner protein requirements:

  • Challenge: atpF functions as part of a complex and may require partner proteins for stability and activity

  • Solution: Co-express with minimal interacting partners; purify subcomplexes rather than individual subunits; use chemical cross-linking to stabilize transient interactions

By systematically addressing these challenges, researchers can significantly improve the quality and reliability of experiments with recombinant N. tomentosiformis atpF, enabling more robust structural and functional studies.

How can researchers efficiently optimize conditions for structural studies of recombinant N. tomentosiformis atpF?

Optimizing conditions for structural studies of recombinant N. tomentosiformis atpF requires a systematic approach across multiple techniques:

• Pre-crystallization screening:

  • Thermal stability optimization: Use differential scanning fluorimetry to identify buffer conditions, additives, and ligands that maximize protein stability

  • Monodispersity assessment: Apply dynamic light scattering and analytical size exclusion chromatography to identify conditions promoting homogeneity

  • Limited proteolysis: Identify stable domains resistant to proteolysis that may be more amenable to crystallization

• Cryo-EM sample preparation optimization:

  • Grid type screening: Test multiple grid types (Quantifoil R1.2/1.3, UltrAuFoil, C-flat) with different hole sizes

  • Glow discharge parameters: Optimize time, current, and atmosphere for glow discharge to achieve optimal hydrophilicity

  • Vitrification conditions: Systematically vary blotting time, force, and temperature to achieve optimal ice thickness

  • Sample concentration: Test concentration range (typically 1-5 mg/mL for membrane proteins) to optimize particle density

• Crystallization condition matrix:

  • Detergent screening: Test multiple detergents and detergent mixtures for their ability to support crystal formation

  • Lipid addition: Supplement with specific lipids (POPC, POPE, cardiolipin) known to stabilize membrane proteins

  • Additive screening: Implement small molecule additives that promote specific crystal contacts

• Protein engineering for structural studies:

  • Construct optimization: Create truncations to remove flexible regions that hinder crystallization

  • Surface entropy reduction: Mutate clusters of high-entropy surface residues (Lys, Glu) to alanine

  • Fusion protein approach: Insert well-folded, crystallizable proteins (T4 lysozyme, rubredoxin) into loops to facilitate crystal contacts

Table 9. Optimization Matrix for Structural Studies of atpF

• Data collection optimization:

  • For X-ray crystallography: Implement grid screening to identify the best diffracting region of crystals; test multiple cryoprotectants; optimize data collection strategy (oscillation range, exposure time)

  • For Cryo-EM: Optimize beam conditions (dose, dose rate); implement beam-shift strategies to collect multiple exposures per hole; test different motion correction and CTF estimation parameters

• Integration of multiple structural techniques:

  • Combine lower-resolution cryo-EM data with X-ray crystallography of individual domains

  • Use solid-state NMR to obtain distance restraints for specific regions of interest

  • Implement hybrid modeling approaches that integrate data from multiple experimental sources

By systematically optimizing these parameters and integrating multiple structural approaches, researchers can overcome the challenges inherent in structural studies of membrane proteins like N. tomentosiformis atpF.

What emerging technologies might enhance our understanding of N. tomentosiformis atpF structure and function?

Several cutting-edge technologies are poised to revolutionize research on N. tomentosiformis atpF:

• Advanced cryo-EM methods:

  • Cryo-electron tomography with subtomogram averaging can visualize atpF in its native membrane environment, revealing native organization and interactions

  • Time-resolved cryo-EM using microfluidic mixing devices captures structural transitions during ATP synthesis

  • Cryo-FIB milling combined with cryo-ET allows visualization of ATP synthase within intact chloroplasts

• Integrative structural biology approaches:

  • AlphaFold2 and RoseTTAFold predictions can provide structural models of atpF and its interactions with other ATP synthase components

  • Integrative modeling platforms combine data from multiple experimental sources (cryo-EM, crosslinking MS, SAXS) to generate comprehensive structural models

  • Molecular dynamics simulations at extended timescales reveal functional motions and energy transduction mechanisms

• Advanced spectroscopic techniques:

  • Single-molecule FRET monitors conformational changes during the catalytic cycle

  • EPR spectroscopy with site-directed spin labeling probes specific domain movements

  • Solid-state NMR provides atomic-level insights into membrane protein dynamics

• Genetic and genome editing technologies:

  • CRISPR-Cas9 base editing creates precise modifications in the native atpF gene to test structure-function hypotheses

  • Prime editing enables scarless introduction of specific mutations without double-strand breaks

  • Synthetic biology approaches allow reconstruction of minimal ATP synthase systems with defined components

• Advanced imaging technologies:

  • Super-resolution microscopy techniques (PALM, STORM) visualize ATP synthase organization in thylakoid membranes

  • Correlative light and electron microscopy (CLEM) connects functional states with structural arrangements

  • Atomic force microscopy provides topographical information and measures mechanical properties of ATP synthase complexes

These technologies will enable researchers to address fundamental questions about N. tomentosiformis atpF that remain unanswered, including the mechanism of proton translocation, the species-specific regulatory mechanisms, and the adaptive advantages of specific structural features.

How might insights from N. tomentosiformis atpF research contribute to synthetic biology and biotechnological applications?

Research on N. tomentosiformis atpF has significant implications for synthetic biology and biotechnology:

• Engineered bioenergetic systems:

  • Designer ATP synthases with modified efficiency or regulatory properties for biotechnological applications

  • Hybrid systems combining features from different species to optimize performance under specific conditions

  • Minimal ATP synthase assemblies with reduced complexity for specific applications

• Biomimetic energy conversion:

  • Artificial photosynthetic systems incorporating optimized ATP synthase components for light-driven ATP production

  • Biohybrid devices integrating biological ATP synthase with synthetic light-harvesting systems

  • Bioelectronic interfaces coupling ATP synthase to electrodes for energy conversion

• Biotechnological applications:

  • Biosensors using ATP synthase components to detect inhibitors, uncouplers, or other compounds affecting bioenergetics

  • Drug screening platforms targeting bioenergetic systems for antimicrobial or anticancer applications

  • Bioproduction systems with enhanced energy efficiency through optimized ATP synthase variants

Table 10. Potential Biotechnological Applications of N. tomentosiformis atpF Research

• Agricultural applications:

  • Enhanced crop photosynthesis through optimization of ATP synthase components

  • Stress-resistant variants derived from understanding N. tomentosiformis adaptations

  • Fine-tuned energy metabolism for improved biomass production

• Evolutionary-inspired design:

  • Computational design of novel ATP synthase components based on evolutionary principles observed in Nicotiana species

  • Directed evolution approaches to optimize atpF for specific applications

  • Cross-species chimeras combining advantageous features from multiple plant species

The insights gained from studying N. tomentosiformis atpF not only enhance our fundamental understanding of bioenergetics but also provide blueprints for engineering improved energy conversion systems for biotechnological applications.