Recombinant Spinacia oleracea ATP synthase 28 kDa subunit, mitochondrial

What is the Spinacia oleracea ATP synthase 28 kDa subunit and how was it initially characterized?

The 28 kDa subunit is a component of the F₀ portion of spinach leaf mitochondrial ATP synthase. It was first identified during comprehensive purification and characterization of the complete ATP synthase complex from spinach mitochondria. The protein was characterized through N-terminal amino acid sequence analysis and Western blot techniques using monospecific antibodies against proteins characterized in other sources. The 28 kDa protein specifically crossreacts with antibodies against the subunit of bovine heart ATPase with N-terminal Pro-Val-Pro- sequence, establishing its homology to the F₀b subunit of Escherichia coli F₀F₁ ATP synthase .

The identification occurred within the context of characterizing the complete spinach mitochondrial ATP synthase, which consists of twelve polypeptides in total. Five of these constitute the F₁ portion, while the remaining seven polypeptides (including the 28 kDa subunit) belong to the membrane-embedded F₀ portion .

How does the 28 kDa subunit contribute to ATP synthase function in plant mitochondria?

The 28 kDa subunit (F₀b homolog) plays several critical roles in ATP synthase function:

Unlike simpler bacterial systems, plant mitochondrial ATP synthases must function in coordination with other energy-transducing systems, making structural components like the 28 kDa subunit particularly important for maintaining optimal enzymatic activity under varying cellular conditions.

How does this subunit compare to homologous proteins in other organisms?

The spinach 28 kDa subunit shows significant N-terminal sequence similarity to the bovine heart ATP synthase subunit, particularly at the Pro-Val-Pro motif . This conservation suggests functional importance of this region. Compared to E. coli's F₀b subunit, the spinach protein is larger but maintains similar structural features that are essential for forming the stator connection between F₀ and F₁ components.

In Euglena gracilis, which has a highly divergent ATP synthase structure, the corresponding subunit shows more variation, reflecting the adaptation of ATP synthase architecture across evolutionary lineages . Despite these differences, the core function of connecting the membrane domain to the catalytic domain is preserved across species.

What are the optimal conditions for recombinant expression of the spinach 28 kDa subunit?

For optimal recombinant expression of the spinach 28 kDa ATP synthase subunit, researchers should consider the following methodological approaches:

Expression System Selection:

• E. coli BL21(DE3) has proven effective for ATP synthase subunit expression, as demonstrated with the epsilon subunit of chloroplast ATP synthase . This strain minimizes proteolytic degradation while maximizing protein yield.

• Alternative hosts such as yeast expression systems may be considered for proteins that require eukaryotic post-translational modifications.

Expression Vector Considerations:

• Include a 6xHis or other affinity tag for simplified purification, preferably with a cleavable linker to remove the tag after purification.

• Use inducible promoters (T7 or tac) with tight regulation to control expression levels.

• Codon optimization may be necessary since plant and bacterial codon usage differs significantly.

Culture Conditions:

• Temperature: Lower temperatures (16-20°C) often improve proper folding of mitochondrial proteins.

• Induction: Use IPTG at 0.1-0.5 mM concentration.

• Media: Enriched media (2xYT or TB) typically yield higher biomass and protein expression.

• Growth phase: Induce at mid-log phase (OD₆₀₀ = 0.6-0.8) for optimal balance between cell density and protein production capacity.

Solubilization Strategy:
Since the 28 kDa subunit is a membrane-associated protein, special consideration should be given to solubilization methods. Based on successful approaches with other ATP synthase subunits, a urea-based protocol may be effective:

• Initial solubilization in 8 M urea

• Controlled dilution into buffer containing ethanol and glycerol to achieve proper folding

This approach has been successfully used for the epsilon subunit of chloroplast ATP synthase from spinach and could be adapted for the mitochondrial 28 kDa subunit.

What purification strategies yield the highest purity and functional integrity of the recombinant protein?

A multi-step purification strategy is recommended to obtain high-purity, functionally active recombinant 28 kDa subunit:

Step 1: Initial Capture

• Immobilized Metal Affinity Chromatography (IMAC) using Ni-NTA for His-tagged proteins

• Wash extensively with low imidazole concentrations (10-20 mM) to remove non-specific binding

• Elute with an imidazole gradient (50-300 mM)

Step 2: Intermediate Purification

• Ion Exchange Chromatography – Based on the predicted pI of the protein

• For the 28 kDa subunit, anion exchange (Q-Sepharose) may be suitable at neutral pH

Step 3: Polishing

• Size Exclusion Chromatography to remove aggregates and achieve highly homogeneous preparations

• Use of a Superdex 75 or similar matrix appropriate for proteins in the 10-100 kDa range

Critical Buffer Components:

• Include glycerol (10-15%) to stabilize the protein structure

• Add reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation of sulfhydryl groups

• Maintain physiological ionic strength (150-200 mM NaCl)

• For membrane proteins, consider including mild detergents (0.03-0.1% n-dodecyl-β-D-maltoside) to maintain solubility

Functional Assessment During Purification:

• Monitor ATPase activity using a coupled enzyme assay at each purification step

• Assess protein-protein interaction capability with other ATP synthase subunits

• Verify proper folding through circular dichroism spectroscopy

This purification strategy is based on successful approaches used for other ATP synthase components and should be optimized specifically for the 28 kDa subunit through iterative testing.

How can researchers assess the structural integrity and functional activity of the purified recombinant 28 kDa subunit?

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm) to determine secondary structure content (α-helices, β-sheets)

  • Near-UV CD (250-350 nm) to assess tertiary structure packing

• Thermal Shift Assay:

  • Measure protein stability under different buffer conditions

  • Determine melting temperature (Tm) as a quality control parameter

• Limited Proteolysis:

  • Exposure to proteases (trypsin, chymotrypsin) at controlled ratios

  • Properly folded proteins show resistance to proteolytic cleavage at certain sites

• Size Exclusion Chromatography with Multi-Angle Light Scattering (SEC-MALS):

  • Determine molecular weight and oligomeric state in solution

  • Assess homogeneity of the preparation

Functional Activity Assessment:

• Reconstitution Assays:

  • Incorporate the purified 28 kDa subunit into liposomes or native membrane systems

  • Measure restoration of ATP synthase activity in preparations lacking this subunit

• Binding Assays:

  • Surface Plasmon Resonance (SPR) or Microscale Thermophoresis (MST) to quantify binding to other ATP synthase subunits

  • Co-immunoprecipitation with partner proteins from the stator structure

• Inhibition of ATPase Activity:

  • Similar to how epsilon subunit inhibits ATPase activity, test if the 28 kDa subunit affects the catalytic properties of the F₁ portion

• Proton Conductance Measurement:

  • Evaluate if the subunit contributes to proton impermeability when reconstituted with other F₀ components

  • Use fluorescent pH indicators in liposome-based assays

• Cross-linking Studies:

  • Chemical cross-linking followed by mass spectrometry to validate correct interactions with neighboring subunits

The combination of these structural and functional assays provides comprehensive validation of the recombinant protein's integrity and biological activity.

What structural interactions does the 28 kDa subunit form with lipid components of the mitochondrial membrane?

The 28 kDa subunit of spinach mitochondrial ATP synthase likely forms critical interactions with membrane lipids, particularly cardiolipins, which are important for ATP synthase function and organization. While specific data for the spinach 28 kDa subunit is limited, insights can be drawn from recent high-resolution structures of related ATP synthases:

Key Lipid Interaction Sites:

• Rotor-Stator Interface:

  • Cardiolipin molecules likely bind at the interface between the 28 kDa subunit and the c-ring rotor

  • These lipids may function as molecular lubricants that facilitate rotation while maintaining the proton-tight seal

• Peripheral Membrane Association:

  • The 28 kDa subunit likely contains amphipathic helices that partially embed in the membrane

  • These regions would interact with phospholipid headgroups while hydrophobic residues insert into the lipid bilayer

• Dimer Interface:

  • ATP synthase forms dimers in the mitochondrial membrane, and the 28 kDa subunit may participate in dimer formation

  • Specific lipids, particularly cardiolipins, may stabilize these interactions at the dimer interface

Recent structural studies of mitochondrial ATP synthase from Euglena gracilis revealed 37 native lipids associated with the complex, including cardiolipins at functionally critical locations . These lipids play roles in:

• Proton translocation efficiency

• Stabilizing subunit interactions

• Facilitating proper membrane curvature essential for cristae formation

While the specific lipid-binding sites on the spinach 28 kDa subunit await detailed structural characterization, researchers investigating this protein should consider these lipid interactions as potentially essential for proper folding, stability, and function of the recombinantly expressed protein.

What molecular cloning strategies are most effective for obtaining the gene encoding the 28 kDa subunit?

Optimal Cloning Approaches for the 28 kDa Subunit Gene:

• Source Material Selection:

  • Fresh spinach (Spinacia oleracea) leaves harvested in the morning when ATP synthase gene expression is typically higher

  • Focus on isolating mitochondria first to enrich for mitochondrial transcripts before RNA extraction

  • Commercial spinach varieties show minimal variation in ATP synthase genes, making most varieties suitable

• Gene Identification Strategy:

  • PCR amplification from cDNA using primers designed based on conserved regions

  • Degenerate primers designed from N-terminal protein sequence data of the 28 kDa subunit (Pro-Val-Pro motif)

  • Alternatively, use genome mining approaches if spinach genome data is available

• Primer Design Considerations:

  • Include appropriate restriction sites flanking the coding sequence

  • Add 6-9 extra nucleotides outside restriction sites to ensure efficient enzyme digestion

  • Consider adding a C-terminal or N-terminal tag sequence (His, FLAG, etc.)

• Optimization of PCR Conditions:

  • Use touchdown PCR protocols to improve specificity

  • Include 5-10% DMSO or betaine to reduce secondary structure formation

  • Test multiple polymerases (Q5, Phusion, Pfu) for optimal amplification

  • Design internal sequencing primers every 300-400 bp

• Vector Selection Criteria:

  • Use pET vectors for high-level expression

  • Consider pET-28a(+) which provides N-terminal His- Tag and T7- Tag with thrombin cleavage site

  • For difficult-to-express proteins, consider pMAL or pGEX fusion systems to improve solubility

• Verification Methods:

  • Restriction digest analysis with multiple enzyme combinations

  • Complete DNA sequencing of the insert and junctions

  • Western blot using antibodies against bovine ATP synthase b-subunit to confirm expression

This systematic approach maximizes the likelihood of successful cloning while providing contingency strategies for challenging aspects of the process.

How can researchers troubleshoot common issues in the recombinant expression of the 28 kDa subunit?

Troubleshooting Guide for Recombinant Expression of the 28 kDa Subunit:

When troubleshooting, implement changes systematically and document results carefully. Start with the most likely causes based on protein characteristics and gradually work through the options until optimal expression is achieved.

What advanced analytical techniques provide the most insightful characterization of the 28 kDa subunit?

Advanced Analytical Techniques for Comprehensive Characterization:

• High-Resolution Structural Analysis:

  • Cryo-Electron Microscopy (Cryo-EM): Particularly valuable for membrane proteins and complexes, can reveal how the 28 kDa subunit integrates into the larger ATP synthase structure

  • X-ray Crystallography: For atomic-level resolution of purified subunit structure

  • NMR Spectroscopy: For analyzing dynamics and interaction surfaces, especially suitable for specific domains of the protein

• Mass Spectrometry Applications:

  • Native MS: Analyze intact protein and potential oligomeric states

  • Hydrogen-Deuterium Exchange MS (HDX-MS): Map solvent-accessible regions and conformational changes upon binding to partner subunits

  • Cross-linking MS (XL-MS): Identify interaction interfaces with other ATP synthase components

  • Post-Translational Modification Analysis: Identify potential regulatory modifications

• Interaction Analysis:

  • Surface Plasmon Resonance (SPR): Quantify binding kinetics with other ATP synthase subunits

  • Isothermal Titration Calorimetry (ITC): Measure thermodynamic parameters of protein-protein interactions

  • Microscale Thermophoresis (MST): Analyze interactions in solution with minimal sample consumption

  • Fluorescence Resonance Energy Transfer (FRET): Monitor dynamic interactions in reconstituted systems

• Functional Analysis:

  • ATPase Activity Assays: Measure impact on ATP hydrolysis rates

  • Proton Translocation Assays: Using pH-sensitive fluorescent dyes in reconstituted liposomes

  • Patch-Clamp Electrophysiology: For direct measurement of proton currents through reconstituted complexes

  • Respiratory Control Ratio Measurements: In reconstituted mitochondrial membranes

• Computational Approaches:

  • Molecular Dynamics Simulations: Model the subunit's behavior in a membrane environment

  • Protein-Protein Docking: Predict interaction interfaces with other subunits

  • Evolutionary Analysis: Compare sequences across species to identify conserved functional domains

• Specialty Techniques for Membrane Proteins:

  • Atomic Force Microscopy (AFM): Visualize topography and mechanical properties

  • Lipid Nanodiscs Reconstitution: Study the protein in a native-like membrane environment

  • Solid-State NMR: Characterize structure in a membrane-embedded state

Researchers should select techniques based on their specific research questions, recognizing that a multi-technique approach often provides the most comprehensive characterization of structure-function relationships.

What mutagenesis approaches can identify critical functional residues of the 28 kDa subunit?

Strategic Mutagenesis Approaches for Functional Characterization:

• Alanine-Scanning Mutagenesis:

  • Systematically replace charged and polar residues with alanine

  • Focus on:

    • Predicted membrane-interface regions

    • Conserved residues identified through sequence alignment

    • Potential lipid-binding sites

  • Example methodology: Generate 3-5 alanine mutations at a time in separate constructs, then narrow down to individual residues in regions showing functional effects

• Charge-Reversal Mutagenesis:

  • Convert positively charged residues (Lys, Arg) to negatively charged ones (Glu, Asp) and vice versa

  • Particularly useful for investigating:

    • Subunit-subunit interfaces

    • Potential interactions with phospholipid head groups

  • This approach was successfully used in studies of the epsilon subunit of ATP synthase to identify key functional interfaces

• Domain Swapping and Truncation Analysis:

  • Replace segments with corresponding regions from other species

  • Create systematic N-terminal and C-terminal truncations

  • Essential for mapping:

    • Minimal functional domains

    • Species-specific functional adaptations

  • Results can be compared to the findings with C-terminal truncations of the epsilon subunit, where removing six amino acids significantly affected function

• Cysteine Substitution and Cross-linking:

  • Introduce cysteine residues at predicted interaction interfaces

  • Use oxidative cross-linking or sulfhydryl-specific cross-linkers

  • Map:

    • Proximity relationships with other subunits

    • Conformational changes during ATP synthesis/hydrolysis

• Conservative vs. Non-conservative Substitutions:

  • For key residues, compare effects of conservative (similar physicochemical properties) vs. non-conservative changes

  • Helps distinguish between:

    • Structural roles (where conservative changes have minimal impact)

    • Specific functional roles (where even conservative changes disrupt function)

Functional Assays for Mutant Evaluation:

This systematic mutagenesis approach, combined with appropriate functional assays, provides a powerful framework for dissecting the structure-function relationships of the 28 kDa subunit in the context of the complete ATP synthase complex.

How can the recombinant 28 kDa subunit be used to study ATP synthase assembly in plants?

The recombinant 28 kDa subunit offers a valuable tool for investigating the assembly pathway of plant mitochondrial ATP synthase through several research approaches:

In vitro Assembly Studies:

• Use fluorescently labeled recombinant 28 kDa subunit to track its incorporation into partially assembled ATP synthase complexes

• Combine with other recombinantly expressed subunits to reconstruct assembly intermediates

• Monitor assembly kinetics using techniques such as fluorescence correlation spectroscopy or native gel electrophoresis

Competitive Binding Assays:

• Introduce recombinant 28 kDa subunit into isolated mitochondria with partially assembled ATP synthase complexes

• Assess displacement of endogenous protein and incorporation rates

• Identify assembly factors that facilitate or inhibit incorporation

Assembly Chaperone Identification:

• Use affinity-tagged recombinant 28 kDa subunit as bait in pull-down assays

• Identify interacting proteins that may function as assembly factors

• Verify interactions using reciprocal co-immunoprecipitation and in vitro binding assays

Dominant Negative Approaches:

• Generate mutant versions of the 28 kDa subunit that can incorporate into complexes but disrupt further assembly

• Use these to trap assembly intermediates for structural and compositional analysis

• Map the temporal sequence of subunit addition in the assembly pathway

This approach builds on findings that newly imported proteins in plant mitochondria can be subject to ATP-dependent proteolysis if they fail to assemble properly, suggesting a quality control mechanism for ATP synthase assembly . By manipulating the availability of the 28 kDa subunit, researchers can observe how these quality control mechanisms respond to assembly perturbations.

What insights can comparative studies between chloroplast and mitochondrial ATP synthase subunits provide?

Comparative analysis of the 28 kDa mitochondrial ATP synthase subunit with its chloroplast counterparts offers unique insights into organelle-specific adaptations of energy conversion machinery:

Evolutionary Divergence Analysis:

Functional Regulation Differences:

Chloroplast ATP synthase undergoes light/dark regulation, while mitochondrial ATP synthase responds to cellular energy status. Comparative studies can reveal:

• Organelle-specific regulatory domains

• Differential responses to pH and ion concentrations

• Unique protein-protein interactions governing activity regulation

Protein Import and Assembly:

The recombinant 28 kDa subunit can be used in import assays with both mitochondria and chloroplasts to:

• Compare organelle targeting efficiency

• Identify organelle-specific assembly factors

• Determine if cross-assembly is possible (mitochondrial subunit into chloroplast ATP synthase)

These studies build on research with the epsilon subunit of chloroplast ATP synthase, which has been extensively characterized and can serve as a methodological template for studying the mitochondrial 28 kDa subunit . The epsilon subunit's role in inhibiting ATPase activity and maintaining proton impermeability may have parallels in how the 28 kDa subunit functions in mitochondrial ATP synthase.

How might structural studies of the 28 kDa subunit contribute to understanding mitochondrial disorders?

Structural and functional studies of the spinach 28 kDa ATP synthase subunit can provide valuable insights into human mitochondrial disorders through comparative analysis with homologous human proteins:

Translational Research Potential:

• Structural Conservation Analysis:

  • The spinach 28 kDa subunit corresponds to ATP5F1 (ATP synthase F₀ subunit b) in humans

  • High-resolution structures of the plant protein can serve as templates for modeling human variants associated with disease

  • Identification of conserved functional domains that may be affected in pathogenic mutations

• Mechanism Elucidation:

  • Understanding how the 28 kDa subunit contributes to ATP synthase stability and assembly

  • Insights into how disruptions in this subunit might lead to energy production deficiencies

  • Clarification of the role of specific domains in proton translocation and energy coupling

• Mutation-Function Correlations:

  • Introducing equivalent disease-associated mutations into the plant protein

  • Assess functional consequences in reconstituted systems

  • Extrapolate findings to understand human disease mechanisms

Methodological Approaches:

• Comparative Structural Analysis:

  • Overlay plant and human protein structures to identify conserved regions

  • Map known disease mutations onto these structures

  • Predict functional consequences based on structural context

• Functional Reconstitution:

  • Use the recombinant 28 kDa subunit in reconstituted systems to measure:

    • ATP synthesis efficiency

    • Proton leak rates

    • Complex stability

  • Compare wildtype performance with disease-mimicking mutations

• Interaction Network Mapping:

  • Identify all protein-protein interactions involving the 28 kDa subunit

  • Determine how these interactions are affected by disease-associated mutations

  • Construct comprehensive interaction maps to visualize disease mechanisms

These approaches can contribute to understanding mitochondrial disorders such as ATP synthase deficiency syndromes, which present with symptoms including cardiomyopathy, neurological deficits, and metabolic dysfunction. The plant system provides a controlled experimental platform for studying fundamental mechanisms that may be conserved in human disease contexts.

What are the challenges and solutions for reconstituting functional ATP synthase with recombinant subunits?

Challenges in ATP Synthase Reconstitution:

• Complex Multi-Subunit Assembly:

  • ATP synthase consists of 12 different subunits in spinach mitochondria

  • Correct stoichiometry and sequential assembly are critical

  • Solution: Develop step-wise assembly protocols starting with F₁ components, then gradually incorporating F₀ subunits including the 28 kDa protein

• Membrane Protein Solubility:

  • The 28 kDa subunit and other F₀ components are membrane proteins

  • Maintaining solubility without compromising structure is challenging

  • Solution: Use mild detergents like DDM or specialized systems like nanodiscs, bicelles, or amphipols

• Lipid Requirements:

  • Native lipids, especially cardiolipins, are essential for ATP synthase function

  • Recombinant systems often lack appropriate lipids

  • Solution: Incorporate defined lipid mixtures that mimic the mitochondrial inner membrane, particularly including cardiolipins

• Proton Gradient Establishment:

  • Functional testing requires generation of a proton gradient

  • Difficult to maintain in artificial systems

  • Solution: Use liposome reconstitution with proton pumps or pH jump techniques

• Structural Validation:

  • Confirming correct assembly is technically challenging

  • Solution: Combine negative-stain EM, native mass spectrometry, and cross-linking studies to verify proper complex formation

Methodological Framework for Successful Reconstitution:

• Expression Strategy:

  • Express all subunits separately with appropriate tags

  • Alternatively, use polycistronic expression systems for coordinated production

  • Purify under conditions that maintain native-like structure

• Assembly Protocol:

  • Controlled detergent removal via dialysis or adsorbents

  • Stepwise addition of subunits in physiologically relevant order

  • Inclusion of assembly chaperones identified from native systems

• Functional Validation:

  • ATP synthesis assays using acid-base transition

  • ATP hydrolysis assays with detection of released phosphate

  • Membrane potential measurements using potential-sensitive dyes

• Troubleshooting Strategies:

  • Use partially assembled native complexes as scaffolds

  • Co-expression of multiple subunits to promote co-folding

  • Screen multiple detergent and lipid combinations systematically

This methodological framework addresses the major challenges of reconstituting functional ATP synthase with recombinant subunits, including the 28 kDa subunit, providing a roadmap for researchers studying structure-function relationships in this complex molecular machine.

What are the future research directions for studies involving the recombinant 28 kDa subunit?

Future research involving the recombinant Spinacia oleracea ATP synthase 28 kDa subunit should focus on several promising directions that build upon current understanding while addressing important knowledge gaps: