Recombinant Triticum aestivum ATP synthase subunit c, chloroplastic (atpH)

What is the optimal expression system for recombinant Triticum aestivum ATP synthase subunit c?

For optimal expression of recombinant Triticum aestivum ATP synthase subunit c (atpH), E. coli-based expression systems are typically most effective, particularly BL21(DE3) strains containing pET vector systems with T7 promoters. This approach allows for controlled induction using IPTG, generally at concentrations between 0.1-1.0 mM. Expression optimization requires testing multiple conditions including induction temperature (typically 18-25°C for membrane proteins rather than standard 37°C), induction time (4-16 hours), and media composition. For downstream structural and functional studies, maintaining proper folding of this hydrophobic membrane protein is critical, which may necessitate the addition of detergents or membrane-mimetic systems during purification. Expression levels should be verified using SDS-PAGE and Western blotting with antibodies specific to either the atpH protein or fusion tags.

How does pH affect the stability and activity of recombinant chloroplastic ATP synthase components?

pH significantly influences both stability and enzymatic activity of chloroplastic ATP synthase components, including the c subunit. While the search results don't specifically address the atpH subunit, we can draw parallels from catalase activity in wheat, which shows optimal activity at neutral pH (pH 7.0) with significantly reduced activity below pH 5.0 . For chloroplastic ATP synthase components, maintaining a pH between 7.0-8.0 typically preserves structural integrity during purification and functional assays, reflecting the pH of the chloroplast stroma during active photosynthesis. During storage and handling of recombinant atpH, buffer systems such as Tris-HCl (pH 7.5-8.0) or HEPES (pH 7.0-7.5) are recommended. For activity assays, researchers should consider the physiological pH ranges that occur during light/dark transitions in the chloroplast, as these affect proton gradients that drive ATP synthase activity.

What purification methods yield the highest purity for recombinant atpH protein?

Purification of recombinant atpH protein requires a strategic approach due to its hydrophobic nature as a membrane protein component. The most effective purification protocol typically involves a multi-step approach: (1) Affinity chromatography using His-tagged constructs with Ni-NTA or TALON resins, eluting with an imidazole gradient (50-300 mM); (2) Size exclusion chromatography to separate aggregates and obtain homogeneous protein preparations; and (3) Ion exchange chromatography as a polishing step. Throughout the purification process, maintaining the protein in appropriate detergent-containing buffers (e.g., 0.03-0.1% n-dodecyl-β-D-maltoside or digitonin) is critical to prevent aggregation. Protein purity should be assessed by SDS-PAGE with silver staining, with expectations of >95% purity for structural and functional studies. Western blotting with specific antibodies confirms protein identity. For chloroplastic membrane proteins like atpH, additional considerations include the presence of appropriate lipids to maintain native-like environments.

How does aluminum stress affect the expression and function of chloroplastic ATP synthase in wheat?

Aluminum stress has complex effects on ATP synthase components in wheat, which may include chloroplastic ATP synthase subunit c (atpH). While the search results primarily discuss mitochondrial F1F0-ATPase responses, we can extrapolate some principles. In Al-resistant wheat cultivars such as PT741, mitochondrial F1F0-ATPase activity increases by 1.8× at 20 μM AlCl3 and by 2.1× at 100 μM AlCl3 . This adaptive response likely helps maintain energy balance within cells under stress conditions. For chloroplastic ATP synthase, researchers should investigate: (1) Transcriptional responses of the atpH gene under Al stress using RT-qPCR; (2) Protein expression levels via Western blotting; (3) ATP synthesis capacity of isolated chloroplasts or reconstituted systems containing recombinant components.

The hypothesis that chloroplastic ATP synthase upregulation may serve as a compensatory mechanism under stress conditions warrants investigation, potentially helping maintain required ATP levels when other cellular processes are compromised. Experimental designs should include multiple Al concentrations (0-150 μM), time-course measurements, and comparisons between Al-resistant and Al-sensitive wheat cultivars to identify genotype-specific responses.

What methods can effectively distinguish between native and recombinant atpH protein function in mixed experimental systems?

Distinguishing between native and recombinant atpH protein function in mixed experimental systems requires specialized approaches. The most effective method involves creating recombinant constructs with specific tags or mutations that don't interfere with function but allow differentiation. Options include:

• Epitope tagging: Small tags (FLAG, c-Myc) at non-critical positions

• Fluorescent protein fusions for visualization (if terminal fusions are tolerated)

• Introduction of unique proteolytic sites for selective cleavage

• Isotope labeling of recombinant protein for mass spectrometry differentiation

When measuring ATP synthase activity in mixed systems, selective inhibition approaches can be employed. For example, if the recombinant protein contains mutations conferring resistance to specific inhibitors that affect the native protein, differential activity measurements become possible. Alternatively, immunodepletion of native protein using specific antibodies before activity assays can help isolate the contribution of the recombinant component. Site-directed mutagenesis of non-critical residues can also create spectroscopic differences (e.g., altered tryptophan fluorescence) that allow differentiation during activity measurements.

How do divalent cations influence the assembly and function of recombinant chloroplastic ATP synthase components?

Divalent cations significantly impact both assembly and function of ATP synthase components. From the search results, we can see that various divalent cations affect enzyme activity differently - Mn²⁺ and Fe²⁺ strongly stimulate catalase activity in wheat, while Ca²⁺, Zn²⁺, and Cu²⁺ have more moderate effects . For chloroplastic ATP synthase, Mg²⁺ is particularly critical for function, as it coordinates with ATP during catalysis.

For recombinant atpH, researchers should consider these effects across multiple experimental contexts:

When working with reconstituted systems containing recombinant atpH, buffer optimization should include testing the effect of these cations on protein stability, oligomerization state, and functional parameters. Analytical techniques such as circular dichroism spectroscopy, thermal shift assays, and activity measurements using coupled enzyme assays can help determine optimal ionic conditions for experimental work.

What are the optimal conditions for functional reconstitution of recombinant atpH into liposomes?

The functional reconstitution of recombinant atpH into liposomes requires careful optimization of multiple parameters to achieve physiologically relevant activity. The optimal protocol includes:

• Lipid composition: A mixture reflecting chloroplast membranes - typically 70% phosphatidylcholine, 15% phosphatidylethanolamine, 10% phosphatidylglycerol, and 5% sulfoquinovosyldiacylglycerol (SQDG). The plant-specific lipid SQDG is particularly important for chloroplastic proteins.

• Protein-to-lipid ratio: Starting with 1:100 (w/w) and testing ratios from 1:50 to 1:200 to determine optimal incorporation without protein aggregation.

• Reconstitution method: Detergent dialysis using mild detergents (0.5-1% n-dodecyl-β-D-maltoside) with controlled detergent removal via step dialysis or Bio-Beads.

• Buffer conditions: 20 mM HEPES or Tricine buffer (pH 7.5), containing 100 mM KCl, 5 mM MgCl₂, and optionally 1-2 mM dithiothreitol to maintain reducing conditions.

• Quality control: Freeze-fracture electron microscopy or dynamic light scattering to assess proteoliposome size distribution and homogeneity.

Functional validation should include proton pumping assays using pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) and ATP synthesis/hydrolysis assays. Orientation of incorporated protein can be determined using protease protection assays, ensuring physiologically relevant directional activity. When reconstituting with other ATP synthase subunits, sequential incorporation may yield better results than simultaneous reconstitution of all components.

How can researchers troubleshoot low expression yields of recombinant atpH in bacterial systems?

Low expression yields of recombinant atpH in bacterial systems are a common challenge due to its hydrophobic nature and potential toxicity to host cells. A systematic troubleshooting approach includes:

• Codon optimization: Analyze the atpH sequence for rare codons in E. coli and either optimize the sequence or use specialized strains containing rare tRNA genes (e.g., Rosetta™).

• Expression construct design:

  • Test multiple affinity tags (His6, GST, MBP) and their positions (N- or C-terminal)

  • Include solubility-enhancing fusion partners like MBP or SUMO

  • Consider using specialized vectors with tight promoter control to prevent leaky expression

• Host strain optimization:

  • C41(DE3) or C43(DE3) strains specifically developed for membrane proteins

  • BL21(DE3) pLysS to reduce basal expression levels

  • Lemo21(DE3) for tunable expression levels

• Culture conditions:

  • Reduce growth temperature to 16-20°C after induction

  • Use enriched media (e.g., Terrific Broth) with glycerol supplementation

  • Test auto-induction media to avoid IPTG toxicity

  • Reduce induction levels (0.01-0.1 mM IPTG instead of standard 0.5-1 mM)

• Extraction optimization:

  • Include specialized detergents for membrane protein extraction (CHAPS, Fos-choline)

  • Test multiple lysis methods (sonication, French press, detergent extraction)

Importantly, small-scale expression trials should be conducted systematically, varying one parameter at a time and analyzing results via Western blotting to determine which combination of factors yields the highest expression of soluble, correctly folded protein before scaling up.

What methods can be used to assess the structural integrity of purified recombinant atpH?

Assessing the structural integrity of purified recombinant atpH is critical for ensuring that functional studies reflect physiologically relevant conformations. Multiple complementary approaches should be employed:

• Circular Dichroism (CD) Spectroscopy: The atpH subunit should show characteristic alpha-helical content with minima at 208 and 222 nm. Thermal denaturation studies using CD can establish the thermal stability (Tm) under various buffer conditions.

• Intrinsic Fluorescence Spectroscopy: If tryptophan or tyrosine residues are present, their fluorescence emission spectra can indicate proper folding, with shifts indicating exposure to aqueous environment versus membrane-like environments.

• Limited Proteolysis: Correctly folded membrane proteins show resistance to proteolysis in their transmembrane regions. Time-course digestion with trypsin or chymotrypsin followed by SDS-PAGE analysis can reveal stable fragments corresponding to structured domains.

• Size Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS): This technique determines the oligomeric state and homogeneity of the purified protein.

• Negative Stain Electron Microscopy: Provides low-resolution structural information to confirm proper assembly of the c-ring structure typical of ATP synthase c subunits.

For higher resolution structural assessment, more advanced techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map solvent-accessible regions, while solid-state NMR can provide atomic-level information about specific residues in the membrane environment. The chosen technique should align with downstream experimental goals – functional studies may require only basic structural validation, while mechanistic investigations would benefit from more detailed structural characterization.

How should researchers interpret contradictory results between in vitro and in vivo studies of atpH function?

When faced with contradictory results between in vitro and in vivo studies of atpH function, researchers should employ a systematic analytical framework. First, recognize that such discrepancies are common for membrane proteins like ATP synthase components, which function within complex supramolecular assemblies. The interpretation should consider:

• Experimental context differences:

  • In vitro systems often lack the complete set of interacting proteins present in vivo

  • Lipid environments differ substantially between artificial systems and native membranes

  • Post-translational modifications may be absent in recombinant systems

• Methodological validation:

  • Verify that activity assays appropriately measure the specific function of interest

  • Confirm protein integrity in both systems using multiple orthogonal techniques

  • Assess whether the recombinant protein truly mimics the native state (oligomerization, orientation)

• Bridging approaches:

  • Develop intermediate complexity systems (e.g., isolated chloroplasts with added recombinant components)

  • Use genetic complementation in ATP synthase-deficient mutants to validate recombinant protein function

  • Apply patch-clamp or other single-molecule techniques to isolated complexes

Based on studies with ATP synthases from various sources, contradictions commonly arise from differences in proton gradient formation, differential effects of regulatory factors, or assembly state differences. For wheat atpH specifically, the wheat cultivar should be considered, as the research results show significant cultivar-specific differences in ATP synthase responses to environmental conditions like aluminum stress . A comprehensive model that accounts for both in vitro and in vivo observations should be developed, potentially revealing regulatory mechanisms that only operate in the cellular context.

What statistical approaches are most appropriate for analyzing the effects of environmental stressors on atpH expression and function?

When analyzing the effects of environmental stressors on atpH expression and function, researchers should select statistical approaches that account for the complex, multi-parameter nature of these experiments. Based on approaches used in the provided search results for similar experiments with wheat proteins , the following statistical framework is recommended:

• Experimental design considerations:

  • Use factorial designs to examine interactions between multiple stressors

  • Include time-course measurements to capture dynamic responses

  • Ensure sufficient biological replicates (minimum n=4) based on observed variability

• For gene expression analysis (RT-qPCR data):

  • Normalize to multiple stable reference genes validated under the specific stress conditions

  • Apply ANOVA followed by appropriate post-hoc tests (Tukey's HSD for all pairwise comparisons)

  • Consider ANCOVA when accounting for covariates like plant developmental stage

• For protein function/activity data:

  • For dose-response relationships, use non-linear regression models (four-parameter logistic)

  • Apply mixed-effects models when incorporating both technical and biological variability

  • Use repeated measures ANOVA for time-course experiments

• Advanced analytical approaches:

  • Principal Component Analysis (PCA) to identify patterns across multiple measured parameters

  • Partial Least Squares Discriminant Analysis (PLS-DA) to identify key variables distinguishing between treatment groups

  • Network analysis when integrating atpH responses with other cellular components

The search results demonstrate these approaches in analyzing ATPase activities across different wheat cultivars under aluminum stress, revealing cultivar-specific responses that wouldn't be apparent with simpler statistical methods . When reporting results, include both relative fold changes and absolute values with appropriate measures of variability (standard deviation or standard error) to facilitate meta-analysis and cross-study comparisons.

How can researchers differentiate between direct effects on atpH and indirect effects through other cellular processes?

Differentiating between direct effects on atpH and indirect effects mediated through other cellular processes requires a multi-faceted experimental approach that systematically isolates variables. This distinction is critical for properly attributing cause and effect in stress response studies such as those examining aluminum toxicity . A comprehensive strategy includes:

• In vitro direct interaction studies:

  • Purified recombinant atpH protein with potential interacting molecules

  • Biophysical methods (isothermal titration calorimetry, surface plasmon resonance) to quantify binding

  • Structural studies (X-ray crystallography, cryo-EM) with and without interacting molecules

• Reconstituted systems of increasing complexity:

  • Proteoliposomes containing only atpH

  • Reconstituted ATP synthase complexes

  • Isolated chloroplasts or chloroplast membranes

• Genetic approaches:

  • Site-directed mutagenesis of potential interaction sites followed by functional testing

  • Complementation studies in atpH-deficient mutants with wild-type vs. modified atpH

  • Inducible expression systems to control timing of atpH expression

• Time-resolved studies:

  • Rapid kinetic measurements (stopped-flow, rapid quench) to establish sequence of events

  • Time-course experiments with closely spaced early time points to capture initial responses

  • Pulse-chase experiments to track modification of existing protein versus synthesis of new protein

A decision tree approach can guide interpretation: If an effect persists in the simplest reconstituted system with purified components, it likely represents a direct effect on atpH. If the effect only appears in more complex systems or whole cells, additional components are likely involved in mediating the response. This approach has successfully distinguished direct effects of aluminum on P-ATPase activity from indirect effects mediated through other cellular signaling pathways in wheat , and can be applied similarly to studies of chloroplastic ATP synthase components.

How might CRISPR-Cas9 genome editing be applied to study atpH function in wheat?

CRISPR-Cas9 genome editing offers powerful opportunities to investigate atpH function in wheat through precise genetic manipulation. A comprehensive research strategy would encompass:

• Gene modification approaches:

  • Complete knockout of atpH to assess essentiality and pleiotropic effects

  • Introduction of point mutations at catalytic sites to create partially functional variants

  • Addition of epitope tags for in vivo tracking without disturbing function

  • Promoter modifications to alter expression levels under specific conditions

• Technical considerations for wheat editing:

  • Design of guide RNAs targeting conserved regions across wheat's three genomes (A, B, and D subgenomes)

  • Optimization of DNA delivery methods for wheat callus (biolistics preferred over Agrobacterium)

  • Selection strategies using both antibiotic resistance and phenotypic screening

  • Verification of edits using deep sequencing to confirm modification across all homeologs

• Physiological analysis of edited plants:

  • Chlorophyll fluorescence measurements to assess photosynthetic efficiency

  • ATP/ADP ratio determination in chloroplasts under various light conditions

  • Growth analysis under normal versus stress conditions (including aluminum stress as highlighted in the search results )

  • Detailed proteomic analysis of ATP synthase complex assembly

• Experimental controls and validation:

  • Complementation with wild-type atpH to confirm phenotype causality

  • Creation of equivalent mutations in model species (Arabidopsis) for comparative analysis

  • Expression of recombinant proteins from edited sequences to confirm biochemical effects

This approach would provide unprecedented insights into structure-function relationships of chloroplastic ATP synthase in its native context and could reveal wheat-specific adaptations not evident in model systems or in vitro studies with recombinant proteins.

What are the most promising approaches for studying interactions between atpH and other ATP synthase subunits?

Studying the interactions between atpH and other ATP synthase subunits requires sophisticated approaches that capture both stable and transient interactions within this complex molecular machine. The most promising methodologies include:

• Structural biology approaches:

  • Cryo-electron microscopy of intact ATP synthase complexes at various catalytic states

  • Cross-linking mass spectrometry (XL-MS) to identify interaction interfaces

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map interaction-induced conformational changes

  • Solid-state NMR of reconstituted complexes in membrane environments

• Protein complementation assays:

  • Split fluorescent protein systems optimized for chloroplast expression

  • Bimolecular Fluorescence Complementation (BiFC) in protoplasts

  • Split luciferase assays for quantitative interaction measurements

  • Förster Resonance Energy Transfer (FRET) between fluorescently labeled subunits

• Computational approaches:

  • Molecular dynamics simulations of the c-ring interacting with other subunits

  • Coevolution analysis to identify co-varying residues across subunits

  • Machine learning models trained on existing structural data to predict novel interaction sites

• Genetic screens:

  • Suppressor mutation analysis to identify compensatory changes in other subunits

  • Synthetic lethality screening to identify functional relationships

  • Directed evolution of optimized subunit interactions

These approaches would build upon observations from the search results showing coordinated responses of different ATPase components under stress conditions , suggesting intricate regulatory interactions between subunits. A multi-technique strategy combining structural insights with functional validation would be most effective, as each method has specific strengths and limitations when applied to membrane protein complexes like ATP synthase.