Recombinant Nandina domestica ATP synthase subunit b, chloroplastic (atpF)

What is ATP synthase subunit b (atpF) and what is its role in chloroplastic energy production?

ATP synthase subunit b (atpF) is a critical component of the chloroplastic F₁F₀-ATP synthase complex, which plays an essential role in energy transduction during photosynthesis. The ATP synthase complex consists of two main parts: the F₁ portion (catalytic domain) and the F₀ portion (membrane-embedded domain). Subunit b functions as part of the peripheral stalk that connects the F₁ and F₀ domains, serving as a critical structural component that prevents rotation of the α₃β₃ hexamer during ATP synthesis.

How does Nandina domestica atpF differ from other plant species?

While specific sequence information for Nandina domestica atpF is not extensively cataloged in the provided resources, comparative analysis with other plant ATP synthase subunits shows species-specific variations. Based on the information available for other ATP synthase subunits from Nandina domestica, such as subunit c (atpH) and subunit a (atpI), we can make some informed inferences.

The atpH subunit from Nandina domestica is 81 amino acids in length with a specific sequence (MNPLISAASVIAAGLAVGLASIGPGVGQGTAAGQAVEGIARQPEAEGKIRGTLLLSLAFMEALTIYGLVVALALLFANPFV) that likely confers species-specific structural and functional properties . Similarly, the atpI subunit is 247 amino acids in length with its unique sequence . These variations suggest that atpF from Nandina domestica would also possess species-specific sequence characteristics that could affect protein-protein interactions within the ATP synthase complex.

What expression systems are most suitable for recombinant production of chloroplastic atpF?

Based on successful expression strategies for other ATP synthase subunits, E. coli expression systems represent the primary choice for recombinant production of chloroplastic atpF. The search results demonstrate that both atpH and atpI from Nandina domestica have been successfully expressed in E. coli systems .

When designing an expression system for atpF, researchers should consider:

• Vector selection: Vectors with strong, inducible promoters such as pET systems for high-level expression, or pMAL systems for fusion protein approaches that may improve solubility .

• Affinity tags: Both atpH and atpI from Nandina domestica have been successfully expressed with His-tags, suggesting this approach would be suitable for atpF as well .

• Codon optimization: Since plant chloroplastic genes may contain codons rarely used in E. coli, codon optimization of the atpF sequence for E. coli expression may improve yields.

• Expression conditions: Optimization of induction conditions (temperature, IPTG concentration, and induction time) is critical for membrane-associated proteins like atpF.

The synthetic gene approach used for spinach ATP synthase subunit c, involving annealed oligonucleotides with appropriate restriction sites, provides a methodological template that could be adapted for Nandina domestica atpF .

What strategies can be employed to improve solubility and yield of recombinant atpF protein?

Recombinant production of membrane-associated proteins like ATP synthase subunit b presents significant challenges due to potential issues with proper folding, solubility, and toxicity to host cells. Several advanced strategies can be implemented to overcome these challenges:

Fusion Protein Approaches:

• MBP (Maltose-Binding Protein) fusion: The pMAL-c2x vector system has been successfully used for other ATP synthase subunits and could be adapted for atpF . MBP can significantly enhance solubility while providing an additional purification handle.

• Thioredoxin fusion: pET-32a(+) vectors incorporate thioredoxin tags that can facilitate proper disulfide bond formation and improve solubility .

• FLAG-tag systems: pFLAG-MAC vectors provide alternatives for proteins that may be problematic with other fusion systems .

Expression Optimization:

• Lower induction temperatures (16-20°C) often improve proper folding of membrane proteins

• Reduced inducer concentrations to slow protein production rate

• Specialized E. coli strains (C41, C43, Rosetta, etc.) engineered for membrane protein expression

• Co-expression with molecular chaperones to assist proper folding

Solubilization Approaches:

• Screening of detergents (mild non-ionic detergents like DDM or LDAO are often effective)

• Addition of specific lipids that may stabilize the protein structure

• Inclusion of osmolytes or stabilizing agents in purification buffers

For Nandina domestica ATP synthase subunits specifically, the successful expression and purification approaches used for subunits atpH and atpI provide valuable templates, with both being expressed as His-tagged proteins in E. coli and purified to >90% purity .

How can researchers verify the structural integrity and functionality of purified recombinant atpF?

Verification of proper folding and functionality is critical for recombinant membrane proteins. For atpF, several complementary approaches should be employed:

Structural Verification:

• Circular Dichroism (CD) spectroscopy to assess secondary structure content

• Size-exclusion chromatography to confirm proper oligomeric state

• Limited proteolysis to examine folding status (properly folded proteins often exhibit distinct proteolytic patterns)

• Thermal shift assays to evaluate protein stability

• NMR or X-ray crystallography for high-resolution structural analysis when feasible

Functional Assessment:

• Reconstitution assays with other ATP synthase subunits to assess proper complex formation

• Binding assays with known interaction partners within the ATP synthase complex

• For full functional assessment, reconstitution into liposomes or nanodiscs followed by ATP synthesis/hydrolysis assays

What are the primary challenges in reconstituting functional ATP synthase complexes using recombinant subunits?

Reconstitution of functional ATP synthase complexes from individual recombinant subunits represents one of the most challenging aspects of this research. Key challenges include:

Assembly Challenges:

• Correct stoichiometric incorporation of multiple subunits

• Proper orientation and insertion of membrane-spanning subunits

• Formation of stable subunit interactions that maintain complex integrity

• Incorporation of essential lipids that may facilitate assembly and function

Functional Verification:

• Development of sensitive assays to measure proton translocation

• Quantification of ATP synthesis/hydrolysis activities

• Assessment of rotational dynamics using specialized biophysical techniques

Current research approaches with spinach c-subunit reconstitution provide valuable methodological insights, focusing on "investigating the factors that influence the stoichiometric variation of the intact ring" . Similar approaches could be applied to Nandina domestica ATP synthase subunits.

A systematic strategy would involve:

• Initial pairwise interaction studies between subunits

• Stepwise addition of subunits to form sub-complexes

• Incorporation into membrane mimetics (nanodiscs, liposomes)

• Functional characterization using complementary biophysical and biochemical assays

What gene synthesis and cloning strategies are recommended for Nandina domestica atpF?

Based on successful approaches with other ATP synthase subunits, the following gene synthesis and cloning strategies are recommended for Nandina domestica atpF:

Gene Synthesis Options:

• Oligonucleotide Assembly: Similar to the approach used for spinach ATP synthase subunit c, where multiple overlapping oligonucleotides (24-46 bp) were annealed and ligated to construct the complete gene . This approach allows for codon optimization and incorporation of desired restriction sites.

• Commercial Gene Synthesis: Services that provide complete synthetic genes with customized codon optimization for E. coli expression.

Cloning Strategy:

The most promising cloning approach based on available data would include:

• Vector Selection: Testing multiple vectors in parallel, including:

  • pMAL-c2x for MBP fusion (enhances solubility)

  • pET vectors for high-level expression with His-tag

  • pFLAG-MAC for alternative fusion options

• Restriction Sites: Designing the synthetic gene with appropriate restriction sites:

  • 5' NdeI and 3' XhoI sites for insertion into pET vectors

  • 5' blunt end and 3' XhoI for insertion into pMAL-c2x at XmnI/XhoI sites

• Affinity Tags: Incorporating N-terminal or C-terminal affinity tags (His-tag being the most commonly used for Nandina domestica ATP synthase subunits)

PCR Amplification Protocol:

• High-fidelity polymerase (such as Phusion) for error-free amplification

• Optimization of annealing temperatures based on primer design

• Addition of restriction sites via primer overhangs

• Verification by sequencing before expression

This comprehensive approach allows for testing multiple expression constructs to identify optimal conditions for recombinant atpF production.

What purification protocols yield the highest purity recombinant atpF protein?

A multi-step purification protocol is recommended for obtaining high-purity recombinant atpF protein, based on successful approaches with other membrane-associated ATP synthase subunits:

Initial Purification Steps:

• Cell Lysis and Membrane Preparation:

  • Mechanical disruption (sonication or high-pressure homogenization)

  • Differential centrifugation to isolate membrane fractions

  • Solubilization using appropriate detergents (screening of multiple options recommended)

• Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA for His-tagged proteins

  • Alternative affinity methods based on fusion partner (amylose resin for MBP fusions)

Secondary Purification Steps:

• Ion Exchange Chromatography:

  • Anion or cation exchange based on theoretical pI of atpF

• Size Exclusion Chromatography:

  • Final polishing step to remove aggregates and achieve homogeneous preparation

Buffer Considerations:

• Inclusion of appropriate detergents throughout purification

• Addition of glycerol (5-50%) to stabilize protein structure

• Storage in Tris/PBS-based buffer with 6% trehalose, pH 8.0, as used for other Nandina domestica ATP synthase subunits

The protocol should aim to achieve >90% purity as determined by SDS-PAGE, similar to what has been reported for recombinant atpH and atpI from Nandina domestica .

How can researchers troubleshoot common expression and purification issues with recombinant atpF?

Researchers working with recombinant membrane proteins like atpF frequently encounter several challenges. Here's a systematic troubleshooting guide:

Low Expression Levels:

• Verify correct reading frame and sequence integrity

• Test alternative expression vectors with different promoter strengths

• Optimize induction conditions (temperature, IPTG concentration, induction time)

• Consider specialized E. coli strains designed for membrane protein expression

• Examine codon usage and optimize if necessary

Protein Insolubility/Inclusion Bodies:

• Reduce induction temperature (16-20°C)

• Decrease inducer concentration

• Test fusion partners known to enhance solubility (MBP, thioredoxin)

• Screen multiple detergents for effective solubilization

• Consider refolding protocols if inclusion bodies are unavoidable

Purification Issues:

• Optimize detergent concentration in purification buffers

• Add stabilizing agents (glycerol, specific lipids, trehalose)

• Include protease inhibitors to prevent degradation

• Adjust imidazole concentrations in IMAC buffers to reduce non-specific binding

• Consider on-column refolding for proteins in inclusion bodies

Storage Stability:

• Test buffer conditions systematically (pH, salt concentration, additives)

• Add glycerol (5-50%) as used for other Nandina domestica ATP synthase subunits

• Store at -20°C/-80°C with aliquoting to avoid freeze-thaw cycles

• Consider lyophilization with appropriate excipients for long-term storage

This methodical approach to troubleshooting, combined with careful documentation of conditions tested, will help researchers optimize recombinant atpF production.

How does recombinant atpF compare structurally and functionally to the native protein?

Assessing the equivalence of recombinant atpF to its native counterpart is crucial for validating experimental findings. Several analytical approaches can be employed:

Structural Comparison:

• Mass spectrometry to confirm exact molecular weight and post-translational modifications

• Circular dichroism to compare secondary structure profiles

• Limited proteolysis patterns to assess tertiary structure similarities

• NMR or X-ray crystallography for high-resolution structural comparison when feasible

Functional Comparison:

• Binding assays with interaction partners from the ATP synthase complex

• Reconstitution experiments comparing complex formation efficiency

• Functional assays measuring contribution to ATP synthesis activity

Potential Differences to Consider:

• Absence of post-translational modifications in bacterial expression systems

• Effects of affinity tags on protein structure and function

• Altered lipid environment affecting protein conformation

• Differences in folding pathway between chloroplast and recombinant systems

For Nandina domestica ATP synthase subunits, successful recombinant expression with high purity (>90%) has been achieved for subunits atpH and atpI , suggesting that similar success could be achieved with atpF using appropriate methodologies.

What molecular biology techniques can be applied to study atpF structure-function relationships?

Recombinant expression systems enable a wide range of molecular biology approaches to investigate structure-function relationships in atpF:

Site-Directed Mutagenesis Studies:

• Systematic mutation of conserved residues to identify functional domains

• Introduction of cysteine residues for cross-linking studies

• Creation of truncation mutants to identify minimal functional domains

Protein Engineering Approaches:

• Domain swapping with homologous proteins from other species

• Introduction of spectroscopic probes at specific positions

• Construction of fusion proteins for FRET-based interaction studies

Biochemical Analysis Techniques:

• Hydrogen-deuterium exchange mass spectrometry to map solvent-accessible regions

• Chemical cross-linking combined with mass spectrometry to identify interaction interfaces

• Surface plasmon resonance to quantify binding kinetics with partner proteins

Biophysical Characterization:

• Single-molecule FRET to study conformational dynamics

• Atomic force microscopy to visualize complex assembly

• Electron microscopy of reconstituted complexes

These techniques, enabled by recombinant protein production, provide powerful tools for dissecting the structural and functional properties of atpF that would be difficult to achieve with native protein purified from plant material.

What are the current research frontiers involving recombinant chloroplastic ATP synthase subunits?

The recombinant production of chloroplastic ATP synthase subunits opens numerous research opportunities at the forefront of photosynthesis research:

Structural Biology Frontiers:

• High-resolution structural determination of complete chloroplastic ATP synthase complexes

• Elucidation of species-specific structural variations in ATP synthase architecture

• Investigation of conformational changes during the catalytic cycle

Functional Investigation Areas:

• Understanding the factors influencing c-ring stoichiometry across species

• Mapping the proton translocation pathway through the membrane domain

• Characterizing the bioenergetics and efficiency of ATP synthesis

Biotechnology Applications:

• Design of modified ATP synthases with altered catalytic properties

• Development of nanomotors based on the rotary mechanism of ATP synthase

• Creation of biosensors utilizing ATP synthase components

Evolutionary Biology Questions:

• Investigation of how ATP synthase structure varies across plant species

• Understanding adaptation of ATP synthase to different environmental conditions

• Comparing chloroplastic, mitochondrial, and bacterial ATP synthases

The ability to produce individual recombinant subunits, as demonstrated with Nandina domestica atpH and atpI , combined with the methodological approaches being developed for reconstitution studies , provides researchers with powerful tools to address these frontier questions in ATP synthase biology.