Recombinant Triticum timopheevii ATP synthase protein MI25

What is Triticum timopheevii ATP synthase protein MI25?

Triticum timopheevii ATP synthase protein MI25 is a mitochondrial protein encoded by the genome of Timopheev's wheat (Triticum timopheevii). The MI25 protein is a component of the ATP synthase complex responsible for ATP production through oxidative phosphorylation. This protein consists of 192 amino acids and is characterized by UniProt ID P68537. It is also known by synonyms including "ATP synthase protein MI25" and "ORF25" . As part of the ATP synthase complex, MI25 contributes to the rotational mechanism that couples proton translocation across the mitochondrial inner membrane with ATP synthesis, a process critical for cellular energy metabolism .

How is recombinant MI25 protein typically produced for research purposes?

The recombinant form of MI25 protein is typically produced using bacterial expression systems, most commonly Escherichia coli. The process involves:

• Cloning the coding sequence into an appropriate expression vector containing an N-terminal His-tag

• Transforming the construct into a competent E. coli strain optimized for protein expression

• Inducing protein expression under controlled conditions

• Cell lysis and purification via nickel affinity chromatography utilizing the His-tag

• Additional purification steps as needed (gel filtration, ion exchange)

• Concentration and lyophilization of the final purified protein

The resulting product is typically supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE .

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

For optimal stability and activity of recombinant MI25 protein, the following storage and reconstitution protocols are recommended:

Before opening, briefly centrifuge the vial to bring contents to the bottom. After reconstitution, the addition of glycerol is recommended for long-term storage, with a typical final concentration of 50% glycerol. Repeated freeze-thaw cycles significantly reduce protein activity and should be avoided .

How can I verify the activity of recombinant MI25 protein in experimental systems?

Verifying the activity of recombinant MI25 protein requires assessing its functional integration into the ATP synthase complex. Methodological approaches include:

• ATPase activity assay: Measure ATP hydrolysis rate spectrophotometrically by coupling ATP hydrolysis to NADH oxidation

• Proton pumping assay: Monitor pH changes or proton flux across membranes containing reconstituted protein

• Membrane potential measurements: Use potential-sensitive fluorescent dyes to assess the protein's contribution to membrane potential

• Co-immunoprecipitation studies: Verify protein-protein interactions with other ATP synthase subunits

• Structural integration analysis: Use blue native PAGE to confirm incorporation into the ATP synthase complex

These functional assays should be complemented with structural verification through Western blotting and mass spectrometry to confirm protein identity and integrity.

How does MI25 protein contribute to mitochondrial function in Triticum timopheevii?

The MI25 protein plays a crucial role in mitochondrial energy production in T. timopheevii. Recent research on ATP synthase complexes indicates that these proteins:

• Facilitate the final step of oxidative phosphorylation by catalyzing ATP synthesis from ADP and inorganic phosphate using the electrochemical gradient of protons across the mitochondrial inner membrane

• Can operate in reverse under certain pathophysiological conditions, hydrolyzing ATP to build the necessary proton gradient across the mitochondrial inner membrane

• Contribute to the maintenance of mitochondrial membrane potential, which is essential for various mitochondrial functions beyond ATP synthesis

• May participate in the formation of the mitochondrial permeability transition pore (mPTP), which regulates cell survival and death signals

In T. timopheevii specifically, the MI25 protein appears to contribute to the unique mitochondrial adaptations that confer stress resistance characteristics to this wheat species . This makes it particularly interesting for comparative studies with other Triticum species that lack equivalent stress tolerance.

What experimental approaches can be used to study the role of MI25 in mitochondrial permeability transition?

Recent evidence suggests that ATP synthase components, including proteins similar to MI25, may participate in forming the mitochondrial permeability transition pore (mPTP). To investigate MI25's potential role in this process, researchers can employ the following approaches:

• Reconstitution studies: Incorporate purified MI25 into artificial lipid bilayers and measure conductance properties

• Mutagenesis experiments: Generate site-specific mutations in MI25 and assess their impact on mPTP formation and mitochondrial function

• Crosslinking and structural studies: Use chemical crosslinking combined with mass spectrometry to identify interaction partners during pore formation

• Functional assays in isolated mitochondria: Measure calcium retention capacity, membrane potential, and swelling in mitochondria with modified MI25 levels

• Imaging techniques: Apply super-resolution microscopy to visualize MI25 localization during mPTP formation

These approaches should be complemented with bioinformatic analyses comparing MI25 with other ATP synthase components implicated in mPTP formation, such as the c-subunit ring structure highlighted in recent literature .

How can hierarchical factor analysis be applied to understand MI25 structure-function relationships?

Hierarchical factor analysis provides a powerful statistical approach for understanding complex relationships between MI25 protein structure and its various functions. This methodology can be applied to:

• Identify patterns in experimental data that correlate specific structural domains with distinct functional properties

• Develop models that account for both direct and indirect relationships between structural features and functional outcomes

• Determine whether observed correlations between structural elements can be explained by a simpler underlying pattern or require a more complex model

For example, researchers could collect data on multiple structural variants of MI25 (through systematic mutagenesis) and measure several functional parameters. Hierarchical factor analysis could then be used to identify which structural features most strongly influence which functions, and whether these relationships can be explained by a simpler underlying model.

When applying this approach, researchers should be careful to:

• Inspect modification indices and use them appropriately to improve model fit

• Consider direct relationships that may not be mediated through the hierarchical structure

• Evaluate multiple models rather than forcing data into an overly simplistic hierarchical structure

What are the key experimental design considerations when studying MI25 protein function?

When designing experiments to investigate MI25 protein function, researchers should consider several methodological factors to ensure valid and reproducible results:

• Control conditions: Implement appropriate negative controls (e.g., heat-inactivated protein) and positive controls (known functional ATP synthase components)

• Experimental validity threats: Address potential threats to internal validity through proper randomization and blinding procedures

• Measurement precision: Ensure consistent measurement techniques with appropriate calibration and technical replicates

• Sample size determination: Conduct power analyses to determine adequate sample sizes for detecting expected effect sizes

• Data analysis planning: Predefine statistical approaches and thresholds for significance before conducting experiments

• Replication strategy: Plan for both technical and biological replication to assess reproducibility

How can I optimize tissue culture conditions for studying MI25 in Triticum timopheevii systems?

When working with T. timopheevii tissue cultures for studying MI25 protein, specific optimization strategies have been found effective:

Early exposure to light significantly increases albino plant development, suppresses somatic embryogenesis, and decreases green plant regeneration. Similarly, elevated copper concentrations have been shown to cause albinism rather than enhance green plant regeneration .

These optimized tissue culture conditions provide a foundation for studying MI25 protein expression and function in its native cellular context, allowing for more physiologically relevant insights than heterologous expression systems alone.

What single-cell analysis approaches can reveal MI25 function in heterogeneous cell populations?

Single-cell analysis techniques offer powerful insights into MI25 protein function across heterogeneous cell populations within T. timopheevii tissues. Key methodological approaches include:

• Single-cell RNA sequencing (scRNA-seq): Reveals MI25 expression patterns and co-expression networks at single-cell resolution using platforms like 10× Genomics

• Spatial transcriptomics: Maps MI25 expression within tissue architecture while preserving spatial information

• Mass cytometry (CyTOF): Quantifies MI25 protein levels alongside numerous other markers in individual cells

• Live-cell imaging: Tracks MI25 dynamics in real-time using fluorescent protein fusions or specific antibodies

Analysis of such data typically involves dimensionality reduction techniques like Uniform Manifold Approximation and Projection (UMAP) to identify cell populations, followed by differential expression analysis to identify cells with unique MI25 expression patterns .

How do genomic approaches inform our understanding of MI25 evolution and function in wheat species?

Recent advances in genomic technologies have expanded our understanding of MI25 evolution and function across wheat species:

• Chromosome-scale genome assembly: The recent chromosome-scale reference genome assembly of T. timopheevii accession PI 94760 based on PacBio sequencing provides a foundation for studying the genomic context of the MI25 gene

• Comparative genomics: Alignment of MI25 sequences across Triticum species reveals evolutionary conservation and divergence patterns that may correlate with functional adaptations

• Transcription factor binding site analysis: Algorithms such as RCisTarget can identify transcription factors that potentially regulate MI25 expression, revealing regulatory networks

• Population genomics: Analysis of MI25 sequence variation across T. timopheevii populations can identify signatures of selection that may indicate functional importance

These genomic approaches provide context for understanding how MI25 function may vary across wheat species and how it contributes to species-specific adaptations.

What emerging technologies hold promise for advanced MI25 protein characterization?

Several cutting-edge technologies are poised to advance our understanding of MI25 protein structure and function:

• Cryo-electron microscopy (Cryo-EM): Enables high-resolution structural determination of MI25 within the ATP synthase complex without crystallization

• AlphaFold and other AI protein structure prediction tools: Provide computational models of MI25 structure that can guide experimental design

• CRISPR/Cas9 genome editing: Allows precise modification of MI25 in its native genomic context to study function

• Single-molecule techniques: Methods such as single-molecule FRET can reveal conformational dynamics during function

• Proteomics approaches: Techniques like hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map protein interactions and conformational changes

• Organoid and tissue engineering: Development of wheat-derived organoids could provide more physiologically relevant systems for studying MI25 function

These technologies collectively offer unprecedented opportunities to understand MI25 function at multiple scales, from atomic structure to organismal physiology.

How might MI25 research contribute to crop improvement strategies?

Understanding the structure and function of MI25 protein has several potential applications in crop improvement:

• Enhanced stress tolerance: If MI25 contributes to T. timopheevii's known stress resistance, this knowledge could be transferred to bread wheat through targeted breeding or genetic engineering

• Improved energy efficiency: Optimization of ATP synthase function could potentially enhance mitochondrial efficiency and crop yield

• Biomarker development: MI25 variants or expression patterns might serve as molecular markers for selecting superior crop varieties

• Pathogen resistance: If MI25 plays a role in programmed cell death through mPTP formation, this pathway might be harnessed for improved pathogen resistance

Integrating findings from MI25 research with the chromosome-scale genome assembly of T. timopheevii provides a foundation for translating basic research into practical crop improvement strategies through precision breeding or genetic engineering approaches.