Recombinant Triticum aestivum ATP synthase protein MI25

What is ATP synthase protein MI25 and what is its role in wheat (Triticum aestivum)?

ATP synthase protein MI25 is a 192-amino acid protein component of the ATP synthase complex in wheat. According to structural analysis, it functions in energy production processes, specifically in adenosine triphosphate synthesis. Recent proteomics studies suggest its additional involvement in phosphate homeostasis and stress response in plants .

The protein has the following specifications:

• Full length: 192 amino acids (1-192)

• UniProt ID: P68538

• Synonyms: ATP synthase protein MI25; ORF25

• Amino acid sequence: MRFLSTDMKDRNMLFAAIPSICASSPKKISIYNEEMIVARCFIGFLIFSRKSLGKTFKET LDGRIESIQEELLQFFNPNEVIPEESNEQQRLLRISLRICSTVVESLPTARCAPKCEKTV QALLCRNLNVKSATLLNATSSRRIRLQDDIVTGFHFSVSERFVSGSTFKASTIDLIREGL IVLRKVRVGGSI

How is recombinant Triticum aestivum ATP synthase protein MI25 typically produced for research applications?

For scientific research, recombinant ATP synthase protein MI25 is typically expressed in Escherichia coli bacterial systems with an N-terminal histidine tag for purification purposes . The expression and purification process involves:

• Gene cloning into an appropriate expression vector

• Transformation into E. coli expression strains

• Induction of protein expression under optimized conditions

• Cell lysis and extraction of the target protein

• Purification via immobilized metal affinity chromatography

• Quality control through SDS-PAGE (ensuring >90% purity)

• Lyophilization for stable storage

The final product is generally supplied as a lyophilized powder that requires proper reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL for experimental use .

What experimental approaches are most effective for studying the structure-function relationship of ATP synthase protein MI25?

To study structure-function relationships of ATP synthase protein MI25, researchers should employ a multi-faceted approach:

Structural Analysis Methods:

• X-ray crystallography or cryo-electron microscopy for high-resolution structure determination

• Circular dichroism spectroscopy to assess secondary structure elements

• Hydrogen-deuterium exchange mass spectrometry to identify flexible regions

• Molecular dynamics simulations to understand conformational changes

Functional Analysis Methods:

• Site-directed mutagenesis of conserved residues followed by activity assays

• Cross-linking studies to identify protein-protein interactions

• Reconstitution in liposomes to measure ATP synthesis activity

• Complementation studies in yeast or plant ATP synthase mutants

Correlating structure with function requires parallel assessment of how structural alterations affect biochemical activities and protein-protein interactions within the ATP synthase complex.

How does ATP synthase protein MI25 contribute to phosphate homeostasis in plants?

Recent proteomics research indicates that ATP synthase protein MI25 plays an important role in plant phosphate homeostasis through the following mechanisms:

• Integration with vacuolar phosphate transport systems: Studies in Brassica napus show that ATP synthase components, including ATP synthase protein MI25, have reduced abundance in vacuolar phosphate transporter mutants (PHT5;1a) .

• Regulation of ATP synthesis capacity: Wild-type plants maintain higher levels of ATP synthase components compared to phosphate transporter mutants, suggesting coordinated regulation between phosphate transport and ATP production .

• Phosphate distribution between compartments: ATP synthase protein MI25 likely influences the distribution of phosphate between storage compartments (vacuoles) and energy-generating organelles (mitochondria/chloroplasts) .

• Response to phosphate limitation: Under phosphate deficiency, plants must balance phosphate allocation between storage and energy production, with ATP synthase components being crucial regulatory targets .

Comparative proteomics has revealed that almost all ATP synthase components, including ATP synthase protein MI25 (BnaCnng12890D), showed increased abundance in wild-type plants compared to vacuolar phosphate transporter mutants .

What are the optimal conditions for reconstitution and storage of recombinant ATP synthase protein MI25?

Based on manufacturer recommendations for recombinant ATP synthase protein MI25, the following protocol ensures optimal reconstitution and storage:

Reconstitution Protocol:

• Centrifuge the vial briefly to bring contents to the bottom before opening

• Reconstitute the lyophilized protein in deionized sterile water to achieve 0.1-1.0 mg/mL concentration

• Add glycerol to a final concentration of 5-50% (50% is recommended as the default concentration)

• Aliquot the reconstituted protein to minimize freeze-thaw cycles

Storage Conditions:

Important: Repeated freeze-thaw cycles should be avoided as they can compromise protein integrity and activity .

What analytical techniques are most appropriate for validating the purity and activity of recombinant ATP synthase protein MI25?

For comprehensive quality control of recombinant ATP synthase protein MI25, researchers should employ the following complementary analytical techniques:

Purity Assessment:

• SDS-PAGE with Coomassie or silver staining (>90% purity standard)

• Western blotting using anti-His tag or protein-specific antibodies

• Size exclusion chromatography to detect aggregates or degradation products

• Mass spectrometry for molecular weight confirmation and detection of modifications

Functional Validation:

• ATP hydrolysis assays (reverse reaction is often easier to measure than synthesis)

• Reconstitution into liposomes to measure proton translocation

• Circular dichroism to confirm proper protein folding

• Thermal shift assays to evaluate protein stability

Activity Measurement Approaches:

• Coupled enzyme assays linking ATP production/hydrolysis to NAD(P)H oxidation/reduction

• Bioluminescence assays using luciferase for sensitive ATP detection

• Isothermal titration calorimetry for thermodynamic parameters

• Proton gradient measurements using pH-sensitive fluorescent dyes

How can ATP synthase protein MI25 be employed in studies of plant stress response mechanisms?

ATP synthase protein MI25 serves as an excellent molecular probe for investigating plant stress responses through several research approaches:

• Comparative Proteomics Under Stress Conditions:

  • Quantify changes in ATP synthase protein MI25 abundance under various stresses (salinity, drought, phosphate limitation)

  • Compare responses across different plant tissues and developmental stages

  • Integrate with transcriptomics to identify regulatory mechanisms

• Stress Response Pathway Mapping:

  • Use ATP synthase protein MI25 as a marker for energy metabolism adjustments

  • Study co-regulation with other stress-responsive proteins

  • Identify signaling pathways connecting stress perception to energy production changes

• Engineering Applications:

  • Overexpress or modify ATP synthase protein MI25 to enhance stress tolerance

  • Develop transgenic lines with altered expression for phenotypic analysis

  • Create reporter constructs to monitor ATP synthase regulation under stress

• Methodological Approaches:

  • Immunoprecipitation to identify stress-induced protein interactions

  • Chromatin immunoprecipitation to study transcriptional regulation

  • Phosphoproteomics to detect post-translational modifications under stress

Recent research in wheat exposed to high salinity (200 mM NaCl) for 15 days revealed significant proteome adjustments in energy metabolism proteins , suggesting that ATP synthase components may serve as important markers for understanding stress adaptation mechanisms.

What insights can comparative analysis of ATP synthase protein MI25 across different plant species provide?

Comparative analysis of ATP synthase protein MI25 across plant species offers valuable evolutionary and functional insights:

Evolutionary Conservation Analysis:

• Sequence homology assessment reveals conserved functional domains

• Identification of species-specific adaptations in energy metabolism

• Phylogenetic analysis to trace the evolutionary history of ATP synthase components

Functional Divergence Exploration:

• Comparison of expression patterns under similar stress conditions

• Analysis of post-translational modifications across species

• Investigation of protein-protein interaction networks in different plants

Methodological Approach:

• Multiple sequence alignment of ATP synthase protein MI25 sequences

• Homology modeling based on available structures

• Heterologous expression of variants from different species

• Cross-species complementation studies in mutant backgrounds

Recent proteomics research comparing ATP synthase components between Brassica napus and other plants identified critical roles in phosphate homeostasis , suggesting conserved functions across species while highlighting specific adaptations to different ecological niches.

What are common challenges in working with recombinant ATP synthase protein MI25 and how can they be addressed?

Researchers working with recombinant ATP synthase protein MI25 frequently encounter several challenges that can be systematically addressed:

Challenge 1: Protein Solubility and Stability Issues

• Solution: Optimize buffer conditions (pH 8.0 Tris/PBS buffer with 6% Trehalose has proven effective)

• Alternative: Express with solubility-enhancing fusion partners (MBP, SUMO)

• Prevention: Store with 50% glycerol at -80°C in small aliquots to prevent freeze-thaw damage

Challenge 2: Functional Activity Loss During Purification

• Solution: Utilize gentle purification methods with detergents that maintain native-like membrane environment

• Alternative: Consider on-column refolding techniques

• Prevention: Minimize time between cell lysis and final purification steps

Challenge 3: Non-Specific Binding in Interaction Studies

• Solution: Include appropriate blocking agents and stringent washing steps

• Alternative: Use label-free interaction detection methods

• Prevention: Validate interactions with multiple complementary techniques

Challenge 4: Difficulty in Quantitative Analysis

• Solution: Develop robust standard curves with purified protein for quantification

• Alternative: Use isotope-labeled reference peptides for mass spectrometry quantification

• Prevention: Include internal standards and technical replicates in all analyses

How can researchers interpret contradictory data regarding ATP synthase protein MI25 function in different experimental systems?

When facing contradictory experimental results regarding ATP synthase protein MI25 function, researchers should implement the following analytical framework:

Systematic Analysis Process:

• Methodological Differences Analysis:

  • Compare experimental conditions (pH, temperature, salt concentration)

  • Assess protein preparation methods (tag position, purification approach)

  • Evaluate assay sensitivity and specificity differences

• Biological Context Evaluation:

  • Consider tissue-specific or developmental differences

  • Analyze genotype variations between experimental systems

  • Examine environmental conditions during sample preparation

• Integrated Multi-Omics Approach:

  • Combine proteomics with transcriptomics data

  • Correlate functional assays with structural information

  • Apply systems biology modeling to reconcile divergent observations

• Independent Validation Strategy:

  • Design critical experiments that directly test conflicting hypotheses

  • Use orthogonal techniques to verify key findings

  • Implement genetic complementation studies in defined backgrounds

For example, apparent contradictions in ATP synthase function between different plant species may reflect evolutionary adaptations to specific environmental challenges rather than methodological errors, as suggested by comparative studies of phosphate homeostasis mechanisms .

What emerging technologies hold promise for advancing our understanding of ATP synthase protein MI25 function?

Several cutting-edge technologies are poised to transform research on ATP synthase protein MI25:

Advanced Structural Biology Approaches:

• Cryo-electron tomography for visualizing ATP synthase in native membranes

• Single-molecule FRET to analyze conformational dynamics during catalysis

• Integrative structural biology combining multiple data sources for complete models

Next-Generation Functional Genomics:

• CRISPR-Cas9 base editing for precise mutagenesis of ATP synthase genes

• Optogenetic control of ATP synthase components to manipulate activity

• Single-cell proteomics to detect cell-specific variations in ATP synthase composition

Innovative Biophysical Techniques:

• High-speed atomic force microscopy for real-time monitoring of ATP synthase dynamics

• Nanoscale thermometry to measure localized heat production during ATP synthesis

• Advanced fluorescent probes for visualizing proton gradients with subcellular resolution

Recent proteomics research has already demonstrated the value of quantitative mass spectrometry in understanding ATP synthase function in phosphate homeostasis , suggesting that further technological advances will continue to reveal new aspects of ATP synthase protein MI25 biology.

What research gaps remain in our understanding of ATP synthase protein MI25 regulation and function under environmental stress?

Despite significant progress, several critical knowledge gaps remain in our understanding of ATP synthase protein MI25:

Unresolved Research Questions:

• Regulatory Mechanisms:

  • How is ATP synthase protein MI25 expression regulated at transcriptional and post-transcriptional levels?

  • What signaling pathways coordinate ATP synthase activity with cellular energy demands?

  • Which post-translational modifications affect ATP synthase protein MI25 function under stress?

• Structural Dynamics:

  • How does ATP synthase protein MI25 contribute to the assembly and stability of the complete ATP synthase complex?

  • What conformational changes occur during catalysis and how are they affected by stress conditions?

  • Are there tissue-specific or stress-induced isoforms with altered properties?

• Metabolic Integration:

  • How is ATP synthase protein MI25 function coordinated with other components of energy metabolism?

  • What role does it play in balancing energy production with phosphate homeostasis ?

  • How do different environmental stresses (drought, salinity, temperature) specifically impact its function?

• Evolutionary Aspects:

  • How has ATP synthase protein MI25 evolved across plant species to adapt to different environmental conditions?

  • Are there crop-specific variations that correlate with stress tolerance or yield stability?

  • Can evolutionary insights inform biotechnological approaches to crop improvement?

Recent proteomics studies have begun to address some of these questions, particularly regarding phosphate homeostasis and salinity stress responses , but comprehensive understanding requires integrated multi-disciplinary approaches.

What are the key parameters and reference values for working with recombinant ATP synthase protein MI25?

Based on published research and manufacturer specifications, the following reference values are recommended for working with recombinant ATP synthase protein MI25:

Table 1: Physical and Biochemical Properties of Recombinant ATP synthase protein MI25

Table 2: Experimental Conditions for ATP synthase protein MI25 Analysis

How do experimental conditions affect the measurement of ATP synthase protein MI25 abundance and activity?

Understanding how experimental conditions influence ATP synthase protein MI25 measurements is critical for experimental design and data interpretation:

Table 3: Effect of Experimental Variables on ATP synthase protein MI25 Measurements

Table 4: Comparative ATP synthase protein MI25 Abundance Under Different Conditions