Recombinant Cycas taitungensis ATP synthase subunit b, chloroplastic (atpF)

How is recombinant Cycas taitungensis atpF typically expressed and purified?

Recombinant Cycas taitungensis atpF is typically expressed in E. coli expression systems, similar to other ATP synthase subunits . Based on established protocols, the following methodology is recommended:

• Gene optimization and cloning: The atpF gene sequence is optimized for E. coli codon usage and cloned into an expression vector with an N-terminal His-tag for purification purposes .

• Expression conditions: The recombinant protein is expressed in E. coli strains such as BL21(DE3) or T7 Express lysY/Iq strains at temperatures typically between 18-30°C to maximize protein solubility .

• Purification strategy:

  • Initial purification via immobilized metal affinity chromatography (IMAC) using the His-tag

  • Optional secondary purification via size exclusion or ion exchange chromatography

  • Final product is typically obtained as a lyophilized powder with >90% purity as determined by SDS-PAGE

The recombinant protein is stored in Tris/PBS-based buffer with 6% trehalose at pH 8.0 for optimal stability .

What are the optimal storage conditions for recombinant Cycas taitungensis atpF protein?

For optimal stability and retention of functional properties, the following storage conditions are recommended:

• Long-term storage: Store lyophilized protein at -20°C to -80°C .

• Working solutions: After reconstitution, store at 4°C for up to one week .

• Reconstitution protocol:

  • Briefly centrifuge the vial before opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage

  • Aliquot to avoid repeated freeze-thaw cycles

Repeated freeze-thaw cycles significantly reduce protein stability and should be avoided .

How can I assess the functional activity of recombinant Cycas taitungensis atpF protein in experimental settings?

Assessment of recombinant atpF functionality requires consideration of its role within the ATP synthase complex. While isolated atpF does not possess catalytic activity, its functional integrity can be evaluated through:

• Reconstitution assays: Incorporate the recombinant atpF into liposomes along with other ATP synthase subunits to form functional complexes, then measure ATP synthesis or hydrolysis rates .

• ADP/ATP exchange measurements: Utilize fluorescence-based methods as an alternative to radioactive assays. This approach allows real-time monitoring of nucleotide exchange without radioisotopes .

The following experimental setup can be employed:

For fluorescence-based activity measurements, preload proteoliposomes with 2 mM ADP and monitor ATP uptake using fluorescently labeled ATP analogues or coupled enzyme assays that produce a fluorescent signal proportional to ATP concentration .

What approaches can be used to investigate the interaction between atpF and other ATP synthase subunits in Cycas taitungensis?

Investigating subunit interactions is crucial for understanding ATP synthase assembly and function. Several complementary approaches can be employed:

• Co-immunoprecipitation (Co-IP): Using antibodies against His-tagged atpF to pull down interacting partners, followed by mass spectrometry identification .

• Surface plasmon resonance (SPR): Quantify binding affinities between immobilized atpF and other purified ATP synthase subunits.

• Cross-linking coupled with mass spectrometry: Identify proximity relationships and interaction interfaces between subunits.

• Cryo-electron microscopy: Determine the structural arrangement of atpF within the ATP synthase complex .

• Yeast two-hybrid or bacterial two-hybrid screening: Identify direct protein-protein interactions between atpF and other ATP synthase components.

Research has shown that atpF (subunit b) forms critical interactions with the other peripheral stalk component (subunit b', encoded by ATPG in Chlamydomonas), and disruption of either component prevents ATP synthase accumulation and function .

How do mutations in the atpF gene affect ATP synthase assembly and function in chloroplasts?

Studies in Chlamydomonas reinhardtii have provided valuable insights into the consequences of atpF mutations:

• Frame-shift mutations: Complete knock-out of atpF function prevents ATP synthase assembly and accumulation, demonstrating its essential role in complex formation .

• Impact on other subunits: In atpF mutants, other ATP synthase subunits show reduced accumulation, indicating coordinated biogenesis of the complex .

• Proteolytic degradation: The absence of functional atpF leads to degradation of unassembled ATP synthase subunits by the thylakoid protease FTSH, with AtpH (subunit c) being particularly susceptible to proteolysis .

• Photosynthetic implications: ATP synthase deficiency in atpF mutants results in high light sensitivity due to impaired photosynthetic electron transport and ATP production .

These findings underscore the critical role of atpF in maintaining structural integrity and functional capacity of chloroplast ATP synthase.

What are the most effective methods for recombinant expression of Cycas taitungensis atpF in E. coli?

Optimizing expression of membrane protein components like atpF requires careful consideration of several factors:

• Vector selection: Compare expression levels using different vectors. Published studies show success with:

  • pMAL-c2x for fusion with maltose-binding protein (MBP)

  • pET-32a(+) for thioredoxin fusion tags

  • pFLAG-MAC for FLAG epitope tags

• Co-expression with chaperones: To improve yield and solubility, co-transform with vectors expressing chaperone proteins such as DnaK, DnaJ, and GrpE (e.g., pOFXT7KJE3 plasmid) .

• Induction conditions optimization:

• Codon optimization: Adapt the Cycas taitungensis atpF sequence for E. coli expression, particularly for rare codons that might limit translation efficiency .

How can I troubleshoot poor expression or solubility issues with recombinant Cycas taitungensis atpF?

When facing challenges with expression or solubility of recombinant atpF, consider the following strategies:

• Expression troubleshooting:

  • Verify plasmid sequence integrity through sequencing

  • Test multiple E. coli strains (BL21, C41/C43, Rosetta)

  • Use autoinduction media to achieve gradual protein expression

  • Add glucose (0.5-1%) to tighten regulation of leaky promoters

• Solubility enhancement:

  • Fusion tags: MBP, SUMO, or thioredoxin tags can significantly improve solubility

  • Detergents: Screen detergents (DDM, LDAO, etc.) for membrane protein solubilization

  • Lysis buffer optimization: Include glycerol (5-10%), reduce ionic strength, add stabilizing agents

• Inclusion body recovery: If atpF forms inclusion bodies, develop a refolding protocol:

  • Solubilize inclusion bodies in 8M urea or 6M guanidine hydrochloride

  • Perform step-wise dialysis to remove denaturant

  • Add phospholipids during refolding to facilitate proper membrane protein folding

• Chaperone co-expression: The use of chaperone proteins (DnaK, DnaJ, GrpE) has been demonstrated to substantially increase quantities of recombinant proteins that are toxic or otherwise difficult to produce .

How should I address data that contradicts expectations in ATP synthase functional studies?

When experimental data contradicts hypotheses about atpF function or ATP synthase activity, follow this systematic approach:

• Examining the data:

  • Thoroughly analyze all results to identify specific discrepancies

  • Pay special attention to outliers that may have influenced results

  • Compare findings with published literature on ATP synthase

• Technical validation:

  • Verify protein integrity via SDS-PAGE and Western blotting

  • Confirm activity assay components are functioning properly

  • Rule out instrument calibration issues or reagent degradation

• Alternative hypotheses:

  • Consider if observed activity could be due to contaminating proteins

  • Evaluate if protein modifications (oxidation, deamidation) are affecting function

  • Assess if buffer conditions or lipid composition may be suboptimal

• Experimental redesign:

  • Modify protocols based on identified issues

  • Implement additional controls to validate each step

  • Consider alternative approaches to measure the same parameter

Remember that unexpected results often lead to new discoveries. The peripheral stalk components of ATP synthase (including atpF) were once thought to be merely structural, but research has revealed their important regulatory roles .

What methods are available for analyzing the integration of recombinant atpF into functional ATP synthase complexes?

Assessing proper integration and functionality of recombinant atpF within the ATP synthase complex requires multiple analytical approaches:

• Biochemical analysis:

  • Blue Native PAGE to visualize intact ATP synthase complexes

  • Size exclusion chromatography to analyze complex formation

  • ATP hydrolysis assays using the ATP regenerating system with pyruvate kinase and lactate dehydrogenase

• Structural characterization:

  • Negative stain electron microscopy to visualize reconstituted complexes

  • Cryo-EM for high-resolution structural analysis of the peripheral stalk

  • Hydrogen-deuterium exchange mass spectrometry to probe structural dynamics

• Functional assessment:

  • ATP synthesis measurements in reconstituted proteoliposomes

  • Proton transport assays using pH-sensitive fluorescent dyes

  • Specific inhibitor studies with oligomycin or aurovertin

• Comparative analysis:

  • Compare functional parameters of complexes containing recombinant vs. native atpF

  • Assess impact of mutations or modifications on complex assembly and function

A critical control is to test inhibitor sensitivity; proper incorporation of atpF should result in a complex that displays characteristic responses to ATP synthase inhibitors.

How can engineered variants of Cycas taitungensis atpF contribute to understanding ATP synthase mechanics?

Engineering atpF variants provides powerful tools for investigating structure-function relationships in ATP synthase:

• Site-directed mutagenesis approaches:

  • Target conserved residues at interfaces with other subunits

  • Introduce cysteine residues for site-specific labeling or cross-linking

  • Create chimeric proteins with atpF sequences from other species to identify species-specific functional regions

• Potential structural modifications:

  • Length alterations in the membrane-spanning domain to investigate proton translocation

  • Modifications to stator regions to understand elastic energy storage

  • Introduction of fluorescent protein fusions for real-time imaging studies

• Experimental applications:

  • Single-molecule studies with labeled atpF to observe conformational changes

  • In vitro reconstitution with modified components to assess impact on rotational catalysis

  • Cross-species complementation to identify evolutionarily conserved functional regions

These approaches could reveal new insights into how the peripheral stalk contributes to energy conversion efficiency and regulatory mechanisms of ATP synthase .

What are the comparative features of atpF across different photosynthetic organisms and what can we learn from them?

Evolutionary comparison of atpF across species reveals important insights about ATP synthase adaptation:

• Sequence conservation patterns:

  • Core functional regions show high conservation across photosynthetic organisms

  • Species-specific variations often correlate with environmental adaptations

  • Cycas taitungensis, as a gymnosperm, represents an evolutionary intermediate between algae and angiosperms

• Structural adaptations:

  • Differences in membrane-spanning regions reflect adaptations to various lipid environments

  • The length and composition of connecting domains vary across species, affecting stator flexibility

• Functional implications:

  • Environmental adaptations (temperature, light conditions) correlate with specific sequence features

  • Variations in regulatory regions suggest different control mechanisms across species

Understanding these evolutionary patterns can guide the design of experiments to identify critical functional domains in atpF and reveal adaptations to different photosynthetic environments.