Recombinant Cicer arietinum ATP synthase subunit b, chloroplastic (atpF)

What is Recombinant Cicer arietinum ATP synthase subunit b, chloroplastic (atpF)?

Recombinant Cicer arietinum ATP synthase subunit b, chloroplastic (atpF) is a protein component of the ATP synthase complex derived from chickpea (Cicer arietinum) chloroplasts and produced through recombinant DNA technology. The atpF gene encodes this specific subunit which is integral to the F0 portion of ATP synthase. This recombinant protein allows researchers to study the structure and function of ATP synthase outside of its native environment. The protein is typically expressed in bacterial systems to generate sufficient quantities for biochemical and structural analyses .

ATP synthase subunit b serves as an important structural component that helps connect the F1 catalytic domain with the membrane-embedded F0 domain, thereby participating in the rotational mechanism that couples proton movement to ATP synthesis. Understanding this protein's role provides insights into energy transduction mechanisms in photosynthetic organisms.

How does ATP synthase function in chloroplasts compared to mitochondria?

In chloroplasts, ATP synthase utilizes the proton gradient established by photosynthetic electron transport, while in mitochondria, the gradient results from respiratory chain activity. The chloroplastic ATP synthase, including the b subunit, has evolved specific adaptations for functioning in the unique environment of the thylakoid membrane and coordinating with photosynthetic processes .

What are standard methods for expressing and purifying recombinant atpF protein?

Standard expression and purification of recombinant Cicer arietinum atpF typically follows these methodological steps:

• Expression System Selection: E. coli strains (BL21(DE3), Rosetta) are most commonly used due to their high expression yields and ease of genetic manipulation.

• Vector Construction: The atpF coding sequence is cloned into an expression vector (pET series) with an appropriate tag (His₆, GST, MBP) to facilitate purification.

• Expression Optimization:

  • Temperature: Typically 18-25°C to enhance proper folding

  • Induction: 0.1-0.5 mM IPTG for 4-16 hours

  • Media: Terrific Broth supplemented with appropriate antibiotics

• Cell Lysis Protocol:

  • French Press (as described in ) or sonication in buffer containing:

    • 20-50 mM Tris-HCl or phosphate buffer (pH 7.0-8.0)

    • 100-300 mM NaCl

    • 5-10% glycerol

    • Protease inhibitors

• Purification Strategy:

  • Initial Capture: Ni²⁺-NTA affinity chromatography for His-tagged proteins

  • Washing: Gradient of imidazole (20-50 mM)

  • Elution: Higher imidazole concentration (250-500 mM)

  • Further Purification: Size exclusion chromatography or ion exchange chromatography

• Storage Conditions:

  • Short-term: 4°C in appropriate buffer

  • Long-term: As precipitate in 70% saturated ammonium sulfate at 4°C or flash-frozen in liquid nitrogen with 10% glycerol

The purification protocol can be modified based on the specific experimental requirements and downstream applications.

How do mutations in the DELSEED-loop region affect ATP synthase function?

Mutations in the DELSEED-loop region have significant effects on ATP synthase function, as demonstrated through deletion studies of the β subunit loop in ATP synthase. Research has shown that this helix-turn-helix structure in the C-terminal domain of the β subunit plays a crucial role in coupling catalysis and rotation . The effects of mutations in this region follow a pattern:

These findings suggest that the critical length of the DELSEED-loop is essential for maintaining the coupling between catalysis and rotation, rather than the specific charged residues within the motif. The increased ATP synthesis in charge-modified mutants may result from altered interactions with the ε subunit, potentially shifting equilibrium to the non-inhibitory "down" conformation during ATP synthesis .

What techniques are most effective for studying protein-protein interactions involving chloroplastic atpF?

Several techniques offer complementary approaches for investigating protein-protein interactions involving chloroplastic atpF, each with specific advantages for different research questions:

For chloroplastic atpF specifically, FRET has proven valuable in confirming the accuracy of structural models regarding the length of the DELSEED-loop . For comprehensive analysis, researchers should employ multiple complementary techniques to overcome the limitations of individual methods.

How does the assembly of atpF into functional ATP synthase complexes occur?

The assembly of atpF (ATP synthase subunit b) into functional ATP synthase complexes follows a coordinated process that involves both nuclear and chloroplast-encoded components. The assembly pathway proceeds through several key steps:

• Subunit Synthesis and Import:

  • Nuclear-encoded subunits (including atpF in many cases) are synthesized in the cytosol with transit peptides

  • Import occurs through the TOC/TIC (Translocon of Outer/Inner Chloroplast membrane) machinery

  • Chloroplast-encoded subunits are synthesized on chloroplastic ribosomes

• Membrane Insertion:

  • The b subunit (atpF) is inserted into the thylakoid membrane through the ALB3/OXA1 insertase system

  • This insertion is critical for establishing the correct topology with the hydrophobic N-terminal domain embedded in the membrane and the hydrophilic C-terminal domain extending into the stromal space

• Formation of Subcomplexes:

  • The F₀ sector forms with sequential assembly of c-ring, followed by a and b subunits

  • The F₁ sector assembles separately, beginning with the α₃β₃ hexamer

• Integration of Subcomplexes:

  • The b subunit (atpF) plays a crucial structural role in connecting F₀ and F₁ sectors

  • The stator stalk, containing the b subunit, forms alongside the central stalk (γ, ε subunits)

  • The length of the b subunit, particularly the DELSEED-loop region, is critical for maintaining proper spatial relationships between components

• Activation and Regulation:

  • Assembly is completed with the addition of small regulatory subunits

  • The complex undergoes conformational adjustments to achieve catalytic competence

  • Interactions between the b subunit and the ε subunit contribute to regulation of activity

Disruptions in the assembly process, particularly those affecting the critical length of connecting elements like the DELSEED-loop, can result in complexes that retain ATPase activity but lose ATP synthesis capability, demonstrating the precise structural requirements for complete functionality .

What controls should be included when studying recombinant atpF functionality?

When studying recombinant atpF functionality, a comprehensive set of controls should be included to ensure experimental validity and interpretable results:

Additionally, when studying the effects of mutations on atpF function, it is essential to include multiple mutant types:

• Conservative mutations (maintain charge/size)

• Non-conservative mutations (alter charge/size)

• Deletion mutations of varying lengths to establish structure-function relationships

• Charge modification mutations (e.g., AALSAAA mutant) to distinguish charge effects from structural effects

These controls help distinguish between effects on catalysis, binding, and structural integrity, enabling more accurate interpretation of experimental results.

How can researchers assess the impact of atpF mutations on ATP synthesis and hydrolysis?

Researchers can employ a multi-faceted approach to comprehensively assess the impact of atpF mutations on ATP synthesis and hydrolysis:

• In Vitro Enzymatic Assays:

  • ATP Synthesis Measurement:

    • Reconstitute purified ATP synthase into liposomes

    • Establish proton gradient using acid-base transition or bacteriorhodopsin

    • Measure ATP production using luciferase assay or coupled enzyme systems

    • Compare rates between wild-type and mutant complexes

  • ATP Hydrolysis Measurement:

    • Assess ATPase activity using colorimetric phosphate release assays

    • Determine temperature dependence of activity to identify structural instabilities

    • Measure activity at various ATP concentrations to generate kinetic parameters (Km, Vmax)

• Bioenergetic Measurements:

  • Proton Pumping Assays:

    • Monitor NADH- and ATP-driven H⁺-pumping using pH-sensitive fluorescent probes

    • Compare efficiency and kinetics between wild-type and mutant ATP synthases

    • Analyze monophasic vs. biphasic proton gradient formation patterns

• Nucleotide Binding Studies:

  • Binding Affinity Determination:

    • Measure nucleotide binding to catalytic sites using fluorescent ATP analogs

    • Assess changes in high-affinity vs. low-affinity binding sites

    • Compare binding patterns between wild-type and mutants (e.g., AALSAAA mutant showing normal MgATP binding pattern)

• Structural Impact Assessment:

  • FRET Analysis:

    • Use fluorescence resonance energy transfer to confirm structural models

    • Measure distances between labeled components to detect conformational changes

    • Verify predicted shortening of loops or altered subunit interactions

• In Vivo Functional Assays:

  • Growth Phenotype Analysis:

    • Analyze growth of strains expressing wild-type or mutant ATP synthase under limiting glucose conditions

    • Correlate growth rates with ATP synthesis capability

    • Assess adaptation to different energy sources

What techniques are available for studying the rotation mechanism of ATP synthase incorporating atpF?

Studying the rotation mechanism of ATP synthase incorporating atpF requires specialized techniques that can detect nanoscale molecular motion. Several advanced methodologies are available:

• Single-Molecule Rotation Assays:

  • Fluorescent Actin Filament Rotation:

    • Attach fluorescent actin filaments to the γ-subunit

    • Immobilize F₁ domain on glass surface

    • Visualize rotation using fluorescence microscopy

    • Measure step size and rotation speed under varying ATP concentrations

  • Gold Nanorod Imaging:

    • Attach gold nanorods to rotor subunits

    • Monitor rotation using dark-field microscopy

    • Achieve higher time resolution than fluorescent methods

    • Calculate torque generated during rotation

• FRET-Based Rotation Detection:

  • smFRET (single-molecule FRET):

    • Label stator (including b subunit) and rotor elements with FRET pairs

    • Monitor distance changes during catalytic cycle

    • Detect conformational changes associated with rotation

    • Correlate with catalytic events

• Structural Studies with Locked Rotation States:

  • Cross-linking Approaches:

    • Introduce cysteine residues at strategic positions for cross-linking

    • Lock the enzyme in defined rotational states

    • Compare activity before and after cross-linking

    • Identify critical interactions between atpF and rotating components

• Computational Approaches:

  • Molecular Dynamics Simulations:

    • Model the entire ATP synthase complex including atpF

    • Simulate rotation and conformational changes

    • Predict effects of mutations on rotation mechanics

    • Calculate energy landscapes during rotation cycle

• Magnetic Bead Rotation Assays:

  • Magnetic Tweezer Systems:

    • Attach magnetic beads to rotor components

    • Apply controlled magnetic fields

    • Measure rotation against known torque

    • Determine force-velocity relationships

The critical length of connecting elements like the DELSEED-loop (as identified through deletion studies) directly impacts the rotational mechanism. When this loop is shortened by ~10 Å through deletion mutations, ATP synthesis capability is lost while ATPase activity is retained, demonstrating the precise structural requirements for coupling catalysis to rotation .

How should researchers interpret contradictory results between ATP synthesis and ATP hydrolysis assays?

When researchers encounter contradictory results between ATP synthesis and ATP hydrolysis assays in studies involving atpF, a systematic analytical approach is essential:

• Recognize Natural Asymmetry in the Processes:

  • ATP synthesis and hydrolysis, while reverse processes, involve different rate-limiting steps and regulatory mechanisms

  • Deletion mutations can affect these processes differently, as seen in studies where 10-residue deletion mutants lost ATP synthesis capability but retained ATPase activity

• Examine Structural Coupling Requirements:

  • ATP synthesis requires precise structural coupling between catalytic sites and the proton-translocating machinery

  • The rotation mechanism demands exact spatial relationships between components

  • Shortening of the DELSEED-loop by ~10 Å disrupts this coupling for synthesis but may permit hydrolysis

• Consider Thermodynamic Differences:

  • ATP synthesis works against a concentration gradient (energetically uphill)

  • ATP hydrolysis is energetically favorable

  • Structural imperfections may prevent the enzyme from performing energetically demanding synthesis while still allowing spontaneous hydrolysis

• Analyze Regulatory Interactions:

  • Interactions with regulatory subunits (particularly ε subunit) differ between synthesis and hydrolysis modes

  • Mutations affecting the DELSEED motif may alter these interactions, as seen in the AALSAAA mutant with increased ATP synthesis activity

  • The ε subunit exists in different conformations ("up" and "down") which differentially affect synthesis and hydrolysis

• Evaluate Data Through Multiple Parameters:

• Diagnostic Tests for Reconciliation:

  • Test effect of PMF (proton motive force) on ATP hydrolysis rates

  • Examine effects of ε subunit removal on both activities

  • Assess binding of ATP analogs that cannot be hydrolyzed

  • Determine if contradictions appear under all conditions or only specific ones

Contradictory results often reveal important mechanistic insights rather than experimental failures, highlighting the distinct requirements for forward and reverse reactions in this complex molecular machine.

What bioinformatic approaches can be used to analyze atpF sequence conservation across plant species?

Bioinformatic approaches for analyzing atpF sequence conservation across plant species should incorporate multiple levels of analysis:

• Primary Sequence Analysis:

  • Multiple Sequence Alignment (MSA):

    • Align atpF sequences from diverse plant species using MUSCLE, MAFFT, or Clustal Omega

    • Identify conserved motifs, particularly in functional regions like the DELSEED-loop

    • Calculate conservation scores for each position

  • Phylogenetic Analysis:

    • Construct maximum likelihood or Bayesian phylogenetic trees

    • Compare atpF evolution with species evolution to identify selective pressures

    • Detect lineage-specific adaptations in different plant groups

• Structural Conservation Analysis:

  • Homology Modeling:

    • Generate structural models of Cicer arietinum atpF based on crystal structures

    • Map conservation data onto 3D structures

    • Identify spatially clustered conserved regions that may indicate functional importance

  • Secondary Structure Prediction:

    • Predict secondary structure elements across species

    • Assess conservation of structural features even when sequence diverges

    • Focus on conservation of critical lengths in connecting regions like the DELSEED-loop

• Functional Site Prediction:

  • Coevolution Analysis:

    • Use methods like Statistical Coupling Analysis (SCA) or Direct Coupling Analysis (DCA)

    • Identify co-evolving residue networks suggesting functional connectivity

    • Detect compensatory mutations that maintain critical interactions

  • Binding Site Prediction:

    • Predict interfaces with other ATP synthase subunits

    • Identify conservation patterns at predicted interfaces

    • Detect species-specific interaction modifications

• Comparative Genomics:

  • Synteny Analysis:

    • Compare genomic context of atpF across plant species

    • Identify conservation of gene order and potential co-regulation

    • Detect gene duplications or rearrangements

  • Promoter Region Analysis:

    • Compare regulatory regions of atpF genes

    • Identify conserved transcription factor binding sites

    • Detect lineage-specific regulatory adaptations

• Visualization and Interpretation Approaches:

These approaches can identify regions that maintain high conservation across species (suggesting crucial functional roles) versus regions with higher variability that may contribute to species-specific adaptations in different photosynthetic contexts.

How can researchers effectively compare experimental data from ATP synthase studies across different species?

Effectively comparing experimental data from ATP synthase studies across different species, including those involving Cicer arietinum atpF, requires a structured approach that accounts for biological variation while enabling meaningful comparisons:

• Standardization of Experimental Conditions:

  • Temperature Normalization:

    • Account for physiological temperature differences between species

    • Use relative temperature scales (% of optimal growth temperature)

    • Apply Arrhenius plots to compare activation energies rather than raw rates

  • Buffer Composition Alignment:

    • Adjust for species-specific pH optima and ion requirements

    • Test multiple conditions to identify species-specific versus general effects

    • Report activities under both optimal and standardized conditions

• Normalization Approaches for Quantitative Comparisons:

• Structural-Functional Correlation:

  • Map functional data onto structural features

  • Compare effects of changes in conserved regions (e.g., DELSEED-loop)

  • Use structure-based alignment rather than sequence-only alignment

  • Focus on equivalent positions rather than residue numbers

• Phylogenetic Context Integration:

  • Group experimental data by evolutionary relationship

  • Identify lineage-specific patterns versus universal features

  • Apply phylogenetic correction to statistical analyses

  • Correlate functional differences with evolutionary distance

• Data Visualization and Integration:

  • Use radar plots to compare multiple parameters simultaneously

  • Develop scoring systems that integrate multiple functional measurements

  • Create interactive visualizations that link sequence, structure, and function

  • Apply principal component analysis to identify key variables separating species

• Statistical Approaches for Cross-Species Comparison:

  • Use ANCOVA with species as covariate

  • Apply hierarchical Bayesian models to account for species as random effect

  • Implement phylogenetically independent contrasts for correlation analyses

  • Calculate effect sizes rather than p-values for meaningful comparisons

By implementing these approaches, researchers can distinguish between species-specific adaptations and universal mechanistic principles in ATP synthase function, extracting meaningful comparisons from diverse experimental datasets. This is particularly important when studying evolutionarily distant organisms or when comparing recombinant proteins like Cicer arietinum atpF with model systems where more extensive data may be available .

What are the key considerations for designing experiments with recombinant Cicer arietinum atpF?

When designing experiments with recombinant Cicer arietinum atpF, researchers should prioritize several key considerations to ensure robust and interpretable results:

• Expression and Purification Strategy:

  • Select appropriate expression systems that can produce correctly folded protein

  • Consider the impact of tags on protein function and interaction capabilities

  • Implement rigorous purification protocols with multiple chromatography steps

  • Verify protein integrity through mass spectrometry and N-terminal sequencing

• Experimental Controls and Validations:

  • Include wild-type controls processed identically to mutant proteins

  • Verify complex assembly when studying subunit interactions

  • Implement parallel activity assays (ATP synthesis and hydrolysis)

  • Validate structural predictions through direct measurement techniques like FRET

• Biological Context Preservation:

  • Consider the native lipid environment when studying membrane interactions

  • Reconstruct complete or partial ATP synthase complexes when possible

  • Account for interactions with regulatory subunits such as the ε subunit

  • Maintain physiologically relevant pH, ion concentrations, and temperatures

• Mechanistic Focus:

  • Design experiments to distinguish between effects on:

    • Catalytic capacity (ATP synthesis/hydrolysis)

    • Structural integrity (complex assembly)

    • Energy coupling (proton translocation)

    • Regulatory interactions (especially with the ε subunit)

  • Focus on the critical length requirements of structural elements like the DELSEED-loop

• Comparative Framework:

  • Compare results with well-characterized model systems

  • Consider evolutionary context when interpreting functional differences

  • Relate findings to specific adaptations in Cicer arietinum's photosynthetic systems

  • Establish clear criteria for cross-species comparisons