Recombinant Angiopteris evecta ATP synthase subunit c, chloroplastic (atpH)

What is the genomic context of ATP synthase subunit c in Angiopteris evecta?

The ATP synthase subunit c (atpH) gene is located in the plastid genome of Angiopteris evecta. The complete plastid genome of A. evecta has been sequenced and consists of 153,901 base pairs, with inverted repeats (IRA and IRB) of 21,053 bp each, a large single-copy (LSC) region of 89,709 bp, and a small single-copy (SSC) region of 22,086 bp . The atpH gene is part of the conserved gene repertoire typical of land plant plastid genomes. The genome organization of A. evecta is most similar to that of Psilotum nudum among sequenced plastid genomes, reflecting their evolutionary relationship within the monilophyte clade .

What laboratory methods are used to isolate chloroplasts from A. evecta for protein extraction?

Chloroplast isolation from A. evecta involves tissue homogenization in an organelle isolation solution containing 0.33 M sorbitol, 50 mM HEPES (pH 7.6), 2 mM EDTA, 1 mM MgCl₂, 0.1% BSA, 1% PVP-40, 1.5 M NaCl, and 5 mM β-Mercaptoethanol . The homogenate is filtered through 30 μm nylon mesh to remove debris. Organelles are then stained with DAPI and Mitotracker Green for visualization and quality assessment before further processing . This method ensures the integrity of chloroplasts prior to protein extraction, which is critical for obtaining functional recombinant proteins.

What strategies optimize expression of recombinant A. evecta atpH in heterologous systems?

Optimizing recombinant expression of A. evecta ATP synthase subunit c requires consideration of several factors. First, codon optimization should be performed based on the expression host (bacterial, yeast, or insect cell systems). When using E. coli expression systems, the inclusion of molecular chaperones can improve proper folding of this membrane protein. Expression vectors containing strong inducible promoters (T7, lac, tac) with fine-tuned induction parameters significantly enhance yield.

For membrane proteins like ATP synthase subunit c, expression as fusion proteins with solubility tags (MBP, SUMO, TrxA) followed by targeted cleavage has proven effective. Additionally, lowering induction temperatures (16-20°C) and using specialized E. coli strains (C41(DE3), C43(DE3)) engineered for membrane protein expression can substantially increase functional protein yields. Post-expression purification should employ mild detergents (DDM, LDAO) to maintain protein integrity during extraction from membranes.

How can researchers distinguish between native and recombinant A. evecta ATP synthase subunit c in experimental systems?

Distinguishing between native and recombinant A. evecta ATP synthase subunit c can be accomplished through several analytical approaches. Epitope tagging (His, FLAG, or HA tags) enables specific detection of the recombinant protein via Western blotting with tag-specific antibodies. Mass spectrometry provides definitive identification based on peptide mass fingerprinting, with recombinant versions showing characteristic modifications from affinity tags.

For functional studies, researchers can introduce silent mutations that alter restriction enzyme sites without changing the amino acid sequence, allowing for restriction fragment length polymorphism (RFLP) analysis. Additionally, incorporating isotope labeling (¹⁵N or ¹³C) in the recombinant protein enables distinction through NMR spectroscopy or mass spectrometry. When expressed in heterologous systems, differences in post-translational modifications between native fern chloroplasts and expression systems can also serve as distinguishing markers.

What are the implications of the unique plastid genome rearrangements in A. evecta for ATP synthase function?

The plastid genome of A. evecta contains several rearrangements compared to other plant lineages, including an inversion of approximately 3Kb involving psbD, psbC, and psbZ genes . This inversion is shared with Psilotum, Adiantum, and Equisetum, serving as a phylogenetic marker for the monilophyte clade . While these genomic rearrangements primarily affect intergenic regions rather than gene positions, they may influence gene expression regulation through altered promoter contexts and transcriptional unit organization.

For ATP synthase function specifically, these genomic architectures could affect operon structure and coordinated expression of ATP synthase subunits. Comparative analyses suggest that despite genome rearrangements, the functional conservation of ATP synthase remains strong due to selective pressure on this essential energy-transducing complex. Researchers should consider these evolutionary adaptations when studying regulatory mechanisms of atpH expression and when designing heterologous expression systems that aim to reproduce native regulatory contexts.

What spectroscopic techniques are most effective for structural characterization of recombinant A. evecta ATP synthase subunit c?

Two-dimensional NMR spectroscopy, particularly Heteronuclear Single Quantum Coherence (HSQC), provides powerful insights into protein structure by mapping carbon-hydrogen correlations . For membrane proteins like ATP synthase subunit c, solid-state NMR is particularly valuable as it allows analysis of the protein in a lipid environment that mimics its native membrane context.

Circular dichroism (CD) spectroscopy enables rapid assessment of secondary structure composition (α-helical content), which is crucial for ATP synthase subunit c as it forms a primarily α-helical hairpin structure. For higher-resolution structural analysis, X-ray crystallography of the entire ATP synthase complex or cryo-electron microscopy can reveal how the c-subunit integrates into the rotor ring and interacts with other subunits.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) provides information about solvent accessibility and structural dynamics of different protein regions, yielding insights into functional conformational changes during the catalytic cycle. These complementary techniques together provide a comprehensive structural characterization essential for understanding structure-function relationships.

What are the optimal conditions for functional reconstitution of recombinant A. evecta ATP synthase subunit c?

Functional reconstitution of recombinant A. evecta ATP synthase subunit c requires careful consideration of lipid composition and protein-to-lipid ratios. The optimal protocol involves solubilizing purified protein in mild detergents (0.05-0.1% n-dodecyl β-D-maltoside) followed by controlled reconstitution into liposomes composed of plant chloroplast lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol, phosphatidylglycerol) at a ratio of approximately 1:100 (protein:lipid).

Reconstitution is typically achieved through detergent removal via dialysis against a buffer containing 10 mM HEPES (pH 7.5), 2.5 mM MgCl₂, and 50 mM NaCl over 48 hours with Bio-Beads addition at specific intervals. The resulting proteoliposomes can be verified for proper incorporation using freeze-fracture electron microscopy and functional assays.

For functional assessment, researchers should establish a proton gradient using acid-base transitions or light-driven proton pumps, then measure ATP synthesis activity using luciferase-based luminescence assays. Alternatively, ATP hydrolysis can be monitored through coupled enzyme assays that detect released phosphate or through direct measurement of proton translocation using pH-sensitive fluorescent dyes.

How can site-directed mutagenesis be utilized to investigate the functional domains of A. evecta ATP synthase subunit c?

Site-directed mutagenesis offers powerful insights into structure-function relationships of the ATP synthase subunit c. Key targets for mutation include the conserved carboxyl residue (typically Asp or Glu) in the transmembrane domain that is essential for proton translocation. Substituting this residue with non-protonatable amino acids (Asn, Gln, or Ala) can elucidate its precise role in proton transport mechanisms.

The interface residues between adjacent c-subunits that form the rotor ring can be mutated to investigate oligomerization properties and ring stability. Using cysteine-scanning mutagenesis, researchers can introduce single cysteine residues at various positions followed by selective labeling with fluorescent or spin-labeled probes to monitor conformational changes during the catalytic cycle.

Mutations at the interface between c-subunits and other ATP synthase components (particularly a and b subunits) help map critical interaction surfaces. Cross-linking strategies combined with mutations can capture transient states during rotation. For all mutagenesis experiments, researchers should employ complementary functional assays including ATP synthesis/hydrolysis measurements, proton pumping assays, and growth complementation in ATP synthase-deficient systems to comprehensively characterize the effects of each mutation.

How can liquid chromatography-mass spectrometry be applied to study post-translational modifications of A. evecta ATP synthase subunit c?

Liquid chromatography-mass spectrometry (LC-MS) provides high-resolution analysis of post-translational modifications (PTMs) on ATP synthase subunit c. The methodology begins with protein digestion using specific proteases (trypsin, chymotrypsin, and Asp-N) to generate overlapping peptide fragments that ensure complete sequence coverage . Nano-LC separation coupled with high-resolution mass spectrometers (Orbitrap or Q-TOF) enables detection of modified peptides.

For comprehensive PTM mapping, researchers should employ multiple fragmentation techniques including collision-induced dissociation (CID), electron-transfer dissociation (ETD), and higher-energy collisional dissociation (HCD) to generate complementary fragmentation patterns. Selective ion monitoring (SIM) can be used to target expected modifications with increased sensitivity . Data analysis requires specialized software implementing probability-based matching algorithms and manual validation of MS/MS spectra.

Common PTMs to investigate include phosphorylation of serine/threonine residues that may regulate assembly or activity, acetylation of lysine residues, and methylation events. Quantitative approaches such as stable isotope labeling or label-free quantification can track dynamic changes in modification patterns under different physiological conditions or in response to environmental stresses.

What bioinformatic tools are most useful for comparative analysis of A. evecta ATP synthase subunit c across species?

Comprehensive comparative analysis of ATP synthase subunit c requires a multi-layered bioinformatic approach. Sequence alignment tools (MUSCLE, MAFFT, T-Coffee) provide the foundation for comparing A. evecta atpH with homologs across evolutionary lineages. For phylogenetic analysis, maximum likelihood (RAxML, IQ-TREE) and Bayesian inference methods (MrBayes, BEAST) can reconstruct evolutionary relationships and detect selection pressures acting on specific residues.

Protein structure prediction tools (AlphaFold2, SWISS-MODEL) generate comparative models that visualize structural conservation across species. Transmembrane topology prediction algorithms (TMHMM, Phobius) help identify conserved functional domains within membrane-spanning regions. Coevolutionary analysis techniques (EVcouplings, GREMLIN) detect co-evolving residue pairs that maintain structural and functional integrity across evolutionary time.

For genome-level comparisons, tools like Mauve and Symap enable visualization of syntenic regions and genomic rearrangements in the context of the plastid genome . Database resources including UniProt, PFAM, and specialized chloroplast genome databases provide additional annotation layers. Integration of these analyses through custom scripts or platforms like Galaxy enables researchers to correlate sequence conservation with structural features and functional importance across diverse plant lineages.

What controls should be included when assessing the physiological role of ATP synthase subunit c in A. evecta?

Rigorous experimental design for investigating ATP synthase subunit c function requires comprehensive controls. Positive controls should include well-characterized ATP synthase preparations from model organisms (Arabidopsis, spinach) with established activity parameters. Negative controls must incorporate heat-denatured enzyme preparations and assays performed in the presence of specific ATP synthase inhibitors (oligomycin, venturicidin, DCCD).

When performing genetic manipulations, researchers should implement complementation controls where the native gene is replaced with wild-type recombinant protein to verify function. For RNA interference or CRISPR-based approaches, off-target effects should be controlled through scrambled sequences and rescue experiments. Tissue-specific or inducible expression systems enable temporal control to distinguish direct from indirect effects.

Environmental variables including light intensity, temperature, and nutrient availability must be standardized across experimental and control groups. When studying protein-protein interactions, researchers should include controls for non-specific binding through mutation of key interaction residues. Quantitative RT-PCR studies should utilize multiple reference genes validated for stability under the experimental conditions. These comprehensive controls ensure that observed phenotypes can be specifically attributed to ATP synthase subunit c function.

How can researchers effectively study the integration of recombinant A. evecta ATP synthase subunit c into functional complexes?

Studying integration of recombinant ATP synthase subunit c into functional complexes requires multi-faceted approaches. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) allows visualization of intact ATP synthase complexes and assembly intermediates. When coupled with second-dimension SDS-PAGE, this technique separates individual subunits within each complex state.

Co-immunoprecipitation using antibodies against other ATP synthase subunits can confirm association of the recombinant c-subunit with native complexes. For in vivo visualization, fluorescently tagged versions (ensuring the tag doesn't disrupt function) enable tracking of incorporation using confocal microscopy. Pulse-chase experiments with differentially labeled subunits help determine the kinetics of assembly.

Proximity labeling techniques (BioID, APEX) identify neighboring proteins in the assembled complex. Single-molecule approaches including fluorescence correlation spectroscopy (FCS) and single-molecule FRET provide insights into dynamic assembly processes. Functional reconstitution assays measuring ATP synthesis activity in proteoliposomes containing varied proportions of recombinant and native subunits can quantify the relationship between incorporation efficiency and functional output.

What emerging technologies offer new insights into the structure-function relationship of A. evecta ATP synthase subunit c?

Several cutting-edge technologies are poised to revolutionize our understanding of ATP synthase subunit c. Cryo-electron tomography now enables visualization of ATP synthase complexes in their native membrane environment at sub-nanometer resolution, revealing physiologically relevant conformational states. Single-molecule force spectroscopy techniques (optical tweezers, magnetic tweezers) can directly measure mechanical forces during c-ring rotation, providing unprecedented insights into energy transduction mechanisms.

Time-resolved serial crystallography using X-ray free-electron lasers (XFELs) captures transient structural states during the catalytic cycle at femtosecond time resolution. Integrative structural biology approaches combining multiple data sources (crystallography, NMR, crosslinking-MS, SAXS) through computational modeling generate more complete structural models than any single technique.

Advanced genetic tools including base editors and prime editors enable precise manipulation of specific codons without double-strand breaks, facilitating subtle functional modifications. Synthetic biology approaches using de novo designed ATP synthase components can test fundamental principles of rotary catalysis. These emerging technologies, particularly when applied in combination, promise to resolve longstanding questions about the molecular mechanisms of this essential enzyme complex.