Recombinant Arabidopsis thaliana ATP synthase protein MI25 (AtMg00640)

What is ATP synthase protein MI25 in Arabidopsis thaliana and what is its role in plant metabolism?

ATP synthase protein MI25 (AtMg00640) is a mitochondrial-encoded subunit of the ATP synthase complex in Arabidopsis thaliana. The protein is essential for the proper assembly and function of the ATP synthase complex, which plays a crucial role in oxidative phosphorylation. The ATP synthase complex harnesses the proton gradient across the inner mitochondrial membrane to synthesize ATP, the primary energy currency of cells.

ATP synthase protein MI25 is encoded by the mitochondrial genome (hence the "Mg" in AtMg00640) rather than the nuclear genome. The protein consists of 192 amino acids and is part of the membranous portion of the ATP synthase complex . Its specific role appears to be in maintaining the structural integrity of the complex and potentially participating in the proton channel functionality.

In plant metabolism, mitochondrial ATP synthase is particularly important during non-photosynthetic periods or in non-photosynthetic tissues, where it serves as the primary source of ATP production. Understanding MI25's function provides critical insights into plant bioenergetics and adaptation mechanisms under varying environmental conditions.

What experimental approaches can be used to characterize the interaction of MI25 with other ATP synthase subunits?

Several experimental approaches can be employed to characterize the interactions between ATP synthase protein MI25 and other subunits of the ATP synthase complex:

• Co-immunoprecipitation (Co-IP): This technique can identify protein-protein interactions by using antibodies specific to MI25 or other ATP synthase subunits. After precipitating the target protein, associated proteins are identified through mass spectrometry or Western blotting. For optimal results, use gentle detergents like n-dodecyl-β-D-maltoside (DDM) to maintain native protein interactions .

• Crosslinking studies: Chemical crosslinking combined with mass spectrometry (XL-MS) can capture transient interactions between MI25 and other subunits. Homobifunctional crosslinkers like disuccinimidyl suberate (DSS) or heterobifunctional crosslinkers can be used depending on the amino acid residues being targeted.

• Cryo-electron microscopy (Cryo-EM): This technique provides high-resolution structural information about the entire ATP synthase complex. Modern cryo-EM approaches can achieve resolutions of 2.6-3.9 Å, allowing visualization of subunit interfaces and interaction details .

• Yeast two-hybrid (Y2H) assays: These can screen for potential interaction partners by expressing MI25 as a bait protein and testing against a library of other ATP synthase subunits.

• Blue Native-PAGE: This technique separates protein complexes in their native state and can be followed by a second dimension of SDS-PAGE to identify individual subunits within the complex.

A comprehensive approach would combine multiple techniques. For example, initial screening with Y2H or Co-IP followed by validation using cryo-EM or crosslinking studies provides both qualitative and structural information about protein interactions.

What are the optimal conditions for expressing recombinant Arabidopsis thaliana ATP synthase protein MI25?

The optimal expression of recombinant Arabidopsis thaliana ATP synthase protein MI25 requires careful consideration of the expression system, vector design, and culture conditions:

Expression System Selection:

• E. coli systems: While commonly used for protein expression, E. coli may present challenges for membrane proteins like MI25. BL21(DE3) or C41/C43(DE3) strains are recommended as they are engineered to better tolerate membrane protein expression.

• Yeast systems: Pichia pastoris or Saccharomyces cerevisiae offer eukaryotic post-translational modifications and better membrane protein folding machinery.

• Insect cell systems: Baculovirus-infected insect cells (Sf9 or Hi5) provide superior folding for complex membrane proteins and are often preferred for ATP synthase subunits.

Vector Design Considerations:

• Include a cleavable affinity tag (His6, GST, or MBP) for purification

• Optimize codon usage for the expression host

• Consider including a signal peptide for membrane targeting

• For difficult expressions, fusion with partners like MBP can enhance solubility

Expression Conditions:

• Induce expression at lower temperatures (16-20°C) to promote proper folding

• Use lower inducer concentrations (e.g., 0.1-0.5 mM IPTG for E. coli)

• Extend expression time (overnight to 72 hours) at reduced temperatures

• Include appropriate additives (glycerol 5-10%, specific lipids) to stabilize membrane proteins

Expression Validation:
Monitor expression using Western blotting with anti-His or anti-MI25 antibodies. Verify proper membrane integration through subcellular fractionation before proceeding to large-scale cultures.

What purification strategies yield the highest purity and activity for recombinant ATP synthase protein MI25?

Purification of recombinant ATP synthase protein MI25 requires specialized approaches due to its membrane protein nature. The following multistep purification strategy can yield high purity and maintain protein activity:

Step 1: Membrane Isolation and Solubilization

• Lyse cells using mechanical disruption (e.g., sonication, French press)

• Isolate membrane fractions through differential centrifugation

• Solubilize membranes using mild detergents like n-dodecyl-β-D-maltoside (DDM) at 1-2% concentration

• Maintain pH around 7.0-8.0 during solubilization

Step 2: Affinity Chromatography

• Use Ni-NTA or similar affinity resin for His-tagged MI25

• Apply solubilized protein in buffer containing 0.05-0.1% DDM

• Wash extensively with increasing imidazole concentrations (10-40 mM)

• Elute with 250-300 mM imidazole

Step 3: Secondary Purification

• Size exclusion chromatography (SEC) using Superdex 200 or similar matrix in buffer containing 0.05% DDM

• Optional ion exchange chromatography depending on theoretical pI of the construct

Step 4: Quality Assessment

• Verify purity using SDS-PAGE (>95% purity)

• Confirm identity via Western blotting and mass spectrometry

• Assess structural integrity through circular dichroism

Activity Preservation Measures:

• Maintain 10-20% glycerol in all buffers

• Include 1 mM DTT or 2-5 mM β-mercaptoethanol to prevent oxidation

• Consider adding phospholipids (0.1-0.5 mg/ml) to stabilize the protein

• Store at -20°C in the presence of 50% glycerol or at -80°C for long-term storage

The purified protein should be assessed for ATP hydrolysis activity using colorimetric assays that measure inorganic phosphate release, similar to methods used for other ATP synthase components .

How can researchers verify the functional integrity of purified recombinant ATP synthase protein MI25?

Verifying the functional integrity of purified recombinant ATP synthase protein MI25 requires multiple complementary approaches:

Biochemical Activity Assays:

• ATP Hydrolysis Activity: Measure ATPase activity using colorimetric assays (malachite green or molybdate) to detect inorganic phosphate release. Compare activity with and without oligomycin, a specific inhibitor of ATP synthase, to confirm specificity .

• Reconstitution in Liposomes: Incorporate purified MI25 into liposomes and assess proton translocation using pH-sensitive fluorescent dyes (e.g., ACMA, pyranine).

Structural Integrity Assessment:

• Circular Dichroism (CD) Spectroscopy: Analyze secondary structure content, particularly alpha-helical content expected for membrane proteins.

• Limited Proteolysis: Compare digestion patterns of purified protein with native protein to assess proper folding.

• Thermal Shift Assays: Determine protein stability through differential scanning fluorimetry.

Interaction Validation:

• Size Exclusion Chromatography: Verify that MI25 elutes at the expected molecular weight or forms appropriate complexes with other ATP synthase components.

• Native PAGE: Assess the ability of purified MI25 to incorporate into larger ATP synthase subcomplexes.

• Surface Plasmon Resonance (SPR): Measure binding kinetics with known interaction partners from the ATP synthase complex.

Functional Data Comparison Table:

A comprehensive assessment using multiple techniques provides the most reliable verification of functional integrity. Comparing results to those obtained with native ATP synthase complexes serves as an important benchmark for quality assessment.

How does the assembly of ATP synthase protein MI25 into the complete ATP synthase complex occur in Arabidopsis thylakoids?

The assembly of ATP synthase protein MI25 into the complete ATP synthase complex in Arabidopsis thylakoids involves a coordinated process with multiple assembly factors and discrete steps:

Assembly Pathway:

• Initial c-ring Formation: The process begins with the assembly of the c-ring in the membrane, which requires specific chaperones. CGL160, a thylakoid membrane protein, plays a crucial role in promoting efficient formation of the membranous c-ring in Arabidopsis thaliana .

• MI25 Integration: ATP synthase protein MI25 is integrated into the developing membrane sector (FO) of the complex, likely in coordination with other membrane-embedded subunits.

• F1 Sector Assembly: Simultaneously, the stromal F1 sector assembles with the help of specific chaperones.

• Complex Completion: The N-terminal domain of CGL160 recruits the stromal ATP synthase intermediate coupling factor CF1 to the membrane-embedded components, facilitating the completion of the fully assembled complex .

Key Assembly Factors:

CGL160 is particularly important in this process. Research has demonstrated that:

• The membrane domain of CGL160 is related to bacterial Atp1 proteins

• The N-terminal domain is specific to the green lineage and is required for late steps in ATP synthase assembly

• This domain specifically recruits the stromal ATP synthase intermediate coupling factor CF1

• This assembly step is critical for chloroplast development, ATP synthase activity, and photosynthesis in Arabidopsis thaliana

Assembly Regulation:

The assembly process is coordinated between nuclear-encoded and organelle-encoded subunits. Nuclear-encoded assembly factors facilitate the incorporation of mitochondrially-encoded subunits like MI25 into the growing complex. This coordination involves:

• Regulated translation of the mitochondrial mRNA encoding MI25

• Co-translational membrane insertion

• Interaction with assembly factors that prevent misfolding or aggregation

• Sequential addition of subunits in a defined order

Disruption of this highly coordinated process, particularly the CGL160-mediated recruitment of CF1, can lead to impaired chloroplast development and reduced photosynthetic efficiency .

What experimental approaches can determine the rotational states and conformational changes of ATP synthase containing MI25?

Investigating the rotational states and conformational changes of ATP synthase containing MI25 requires sophisticated biophysical techniques that can capture dynamic molecular movements:

High-Resolution Structural Methods:

• Cryo-Electron Microscopy (Cryo-EM):

  • Modern cryo-EM achieves resolutions of 2.6-3.9 Å for ATP synthase complexes

  • Can identify different rotational states by classifying particles into discrete conformational classes

  • Enables visualization of nucleotide occupancy in catalytic sites

  • Local refinement of F1 and FO domains can improve resolution for specific regions of interest

• Time-Resolved X-ray Crystallography:

  • Captures structural snapshots during catalytic cycle

  • Requires crystallization of the full complex or relevant subcomplexes

Dynamic Measurement Techniques:

• Single-Molecule FRET (smFRET):

  • Label specific residues on MI25 and interacting subunits with fluorophore pairs

  • Monitor distance changes during ATP hydrolysis or synthesis

  • Can track rotational motion in real-time at millisecond resolution

• High-Speed Atomic Force Microscopy (HS-AFM):

  • Directly visualizes conformational changes of the complex in near-native conditions

  • Can achieve sub-second temporal resolution

  • Allows observation of rotational states without crystallization

• Single-Molecule Rotation Assays:

  • Attach fluorescent probes or beads to rotor components

  • Visualize rotation directly using fluorescence microscopy or optical traps

  • Quantify rotation speed, step size, and dwell times in different conditions

Complementary Biophysical Approaches:

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Site-directed spin labeling of MI25 at specific residues

  • Measures distances between labeled sites during conformational changes

  • Especially useful for membrane-embedded regions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps regions of altered solvent accessibility during the catalytic cycle

  • Identifies conformational changes in different nucleotide-bound states

Data Analysis and Integration:

For comprehensive understanding, researchers should combine multiple techniques and integrate the data. For example, cryo-EM has revealed that ATP synthase can exist in multiple rotational states (commonly referred to as states 1, 2, and 3) . These states differ in the positions of the central stalk and the conformations of the catalytic β subunits (βTP, βDP, and βE), which reflect different stages of the catalytic cycle.

How does the function of Arabidopsis thaliana ATP synthase protein MI25 compare to its homologs in other species under varying environmental conditions?

Comparative analysis of Arabidopsis thaliana ATP synthase protein MI25 with its homologs across species reveals adaptations to different environmental conditions and evolutionary specializations:

Cross-Species Functional Comparison:

• Structural Conservation and Divergence:

  • Core functional domains of MI25 show high conservation across plant species

  • N-terminal regions display greater variability, potentially reflecting species-specific regulatory mechanisms

  • Transmembrane topology is generally preserved, but specific residues may differ based on membrane lipid composition adaptations

• Environmental Adaptation Mechanisms:

  • Temperature sensitivity differences: MI25 from thermophilic organisms contains more hydrophobic residues and stronger ionic interactions for stability

  • pH response variations: Plants adapted to acidic soils show modifications in proton-conducting pathways

  • Salt tolerance: Halophytes exhibit MI25 variants with altered surface charge distributions

Comparative Performance Under Stress Conditions:

Evolutionary Significance:

These evolutionary adaptations influence how ATP synthase functions under various environmental conditions:

• Photosynthetic Efficiency: Species-specific variations in MI25 affect the coupling efficiency between proton translocation and ATP synthesis, particularly under fluctuating light conditions

• Stress Response: MI25 variants contribute to differences in how plants maintain bioenergetic homeostasis during environmental stress

• Metabolic Flexibility: Structural differences in MI25 across species correlate with varied abilities to switch between ATP synthesis and hydrolysis modes depending on cellular energy demands

Understanding these comparative aspects provides insights for engineering ATP synthase for improved crop performance under challenging environmental conditions.

What role does the ATP synthase protein MI25 play in mitochondrial permeability transition and plant cell death pathways?

ATP synthase protein MI25's involvement in mitochondrial permeability transition (MPT) and plant cell death pathways represents an emerging area of research with important implications for understanding plant stress responses:

Mitochondrial Permeability Transition in Plants:

Unlike mammalian systems where ATP synthase is well-established as the core component of the mitochondrial permeability transition pore (mPTP), the role of ATP synthase and specifically MI25 in plant MPT is still being elucidated. Current research indicates:

• Channel Formation Properties:

  • ATP synthase can form voltage-dependent channels in lipid bilayers

  • These channels can exhibit brief openings with the channel mainly dwelling in its closed, inactive state

  • Unlike mammalian ATP synthase channels, plant ATP synthase channels may show reduced sensitivity to calcium

• Regulatory Factors:

  • Channel activity can be modulated by pH, membrane potential, and nucleotides

  • Specific interacting proteins may regulate opening and closing

  • Post-translational modifications of MI25 could alter channel properties

Connection to Plant Cell Death Pathways:

The potential involvement of MI25-containing ATP synthase in programmed cell death includes:

• Hypersensitive Response (HR):

  • During pathogen attack, MPT may contribute to localized cell death

  • MI25 structural changes could facilitate formation of transient pores

  • These pores may release pro-apoptotic factors from mitochondria

• Abiotic Stress Responses:

  • Under severe environmental stress, regulated changes in MI25 conformation might contribute to controlled cell death

  • This process may help conserve resources and protect surrounding tissues

• Developmental Cell Death:

  • During leaf senescence or embryo development, changes in ATP synthase assembly involving MI25 could contribute to programmed tissue remodeling

Experimental Evidence and Limitations:

Current research has demonstrated that:

• Purified ATP synthase from various organisms can form channels in lipid bilayers

• These channels can exhibit different properties depending on the source organism

• The channels show sensitivity to classic modulators of the mPTP

• Site-directed mutagenesis of critical MI25 residues

• Electrophysiological studies of purified complexes

• In vivo imaging of mitochondrial permeability in MI25 mutant plants

Understanding MI25's role in these processes could provide new targets for enhancing plant stress tolerance and controlling pathogen-induced cell death.

What are the most effective experimental designs for studying MI25 function in Arabidopsis thaliana?

Designing effective experiments to study MI25 function in Arabidopsis thaliana requires strategic approaches that combine genetic manipulation, biochemical analysis, and physiological assessment:

Genetic Manipulation Approaches:

• CRISPR/Cas9 Gene Editing:

  • Create precise mutations in the MI25 gene (AtMg00640)

  • Target critical residues identified through structural analysis

  • Generate conditional knockouts using inducible systems

• RNA Interference (RNAi):

  • Design constructs targeting MI25 mRNA

  • Use tissue-specific or inducible promoters to control expression

  • Monitor phenotypic effects under different conditions

• Complementation Studies:

  • Express wild-type or mutated versions of MI25 in knockout/knockdown backgrounds

  • Utilize chimeric proteins with domains from different species to identify critical regions

Experimental Framework:

A comprehensive experimental design should follow the framework outlined in experimental research methodology :

• Control Variables: Maintain consistent growth conditions (light intensity, photoperiod, temperature, humidity) across experiments

• Independent Variables: Systematically vary factors such as:

  • MI25 expression levels

  • Specific mutations in MI25

  • Environmental stressors (temperature, light, drought)

• Dependent Variables: Measure multiple outcomes including:

  • ATP synthase assembly and activity

  • Mitochondrial membrane potential

  • Reactive oxygen species (ROS) production

  • Growth parameters and developmental timing

  • Stress tolerance

Experimental Design Types:

Critical Considerations:

When designing experiments :

• Ensure adequate statistical power through appropriate sample sizes

• Include proper controls (positive, negative, and procedural)

• Validate findings using multiple independent approaches

• Consider pleiotropic effects when interpreting phenotypes

• Account for the maternal inheritance of mitochondrial genes when designing crosses

These experimental design approaches provide a robust framework for investigating MI25 function across multiple levels, from molecular interactions to whole-plant phenotypes.

How can researchers effectively isolate and analyze native ATP synthase complexes containing MI25 from Arabidopsis thaliana?

Isolating and analyzing native ATP synthase complexes containing MI25 from Arabidopsis thaliana requires careful consideration of tissue selection, extraction conditions, and analytical techniques:

Sample Preparation and Initial Extraction:

• Tissue Selection and Growth Conditions:

  • Use young, actively growing tissues (e.g., 14-21 day seedlings) for highest ATP synthase content

  • Grow plants under controlled conditions to ensure reproducibility

  • Consider dark-grown plants for higher mitochondrial yield

• Mitochondrial Isolation:

  • Homogenize tissue in isolation buffer (0.3M sucrose, 50mM Tris-HCl pH 7.5, 3mM EDTA, 0.1% BSA)

  • Use differential centrifugation to separate mitochondria (1,500×g → 3,000×g → 12,000×g)

  • Purify mitochondria further using Percoll gradient centrifugation (18%, 23%, 40% Percoll)

ATP Synthase Complex Extraction:

• Gentle Solubilization:

  • Solubilize mitochondrial membranes with n-dodecyl-β-D-maltoside (DDM) at 1-2% w/v

  • Maintain pH at 7.0 to preserve complex integrity

  • Include protease inhibitors (PMSF, leupeptin, pepstatin A)

• Purification Strategy:

  • Use differential centrifugation followed by polyethylene glycol (PEG) precipitation

  • Apply size exclusion chromatography for final purification

  • Alternatively, use immunoprecipitation with antibodies against conserved ATP synthase subunits

Complex Integrity Verification:

• Activity Assays:

  • Measure oligomycin-sensitive ATP hydrolysis activity using colorimetric assays

  • Determine ATP synthesis rates using luciferin/luciferase-based assays

  • Compare activity before and after purification to assess structural integrity

• Complex Analysis:

  • Perform Blue Native PAGE to visualize intact complexes

  • Use second-dimension SDS-PAGE to identify constituent subunits

  • Verify MI25 presence by Western blotting or mass spectrometry

Advanced Analytical Techniques:

• Cryo-EM Analysis:

  • Apply samples to holey carbon grids and vitrify by plunge-freezing

  • Collect images using a transmission electron microscope with a direct electron detector

  • Process data using software like cryoSPARC to achieve high-resolution structures

  • Perform local refinements of F1 and FO domains to improve resolution of specific regions

• Proteomic Analysis:

  • Digest purified complexes with trypsin

  • Analyze peptides using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

  • Identify post-translational modifications on MI25

  • Quantify stoichiometry of complex components

Troubleshooting Common Issues:

By following these protocols, researchers can isolate intact ATP synthase complexes containing MI25 suitable for structural and functional studies.

What are the best approaches for studying the impact of environmental stressors on ATP synthase MI25 function?

Investigating how environmental stressors affect ATP synthase MI25 function requires multifaceted approaches that span from molecular to whole-plant analyses:

Stress Treatment Systems:

• Controlled Growth Environments:

  • Use growth chambers with precise control of temperature, light, and humidity

  • Design gradient stress treatments (e.g., progressive temperature increase) to identify thresholds

  • Implement cycling stress regimes that mimic natural conditions

• Stress Application Methods:

  • Temperature stress: Apply precise temperature regimes (heat: 35-42°C; cold: 4-10°C)

  • Drought stress: Controlled soil moisture deficit or polyethylene glycol (PEG) treatment

  • Salt stress: Defined NaCl concentrations (50-200 mM)

  • Oxidative stress: H₂O₂ or methyl viologen application

  • Nutrient deficiency: Modified Hoagland solutions lacking specific elements

Molecular and Biochemical Analyses:

• Transcriptional Responses:

  • Quantitative RT-PCR for MI25 and related genes

  • RNA-Seq to capture global transcriptional changes

  • Analysis of transcript processing and stability

• Protein Dynamics:

  • Quantify MI25 protein levels via Western blotting

  • Assess post-translational modifications using mass spectrometry

  • Examine protein turnover rates with pulse-chase experiments

• ATP Synthase Function:

  • Measure ATP synthase activity in isolated mitochondria

  • Monitor proton translocation efficiency

  • Determine ATP synthesis rates under different stress conditions

Physiological and Cellular Analyses:

• Mitochondrial Function:

  • Measure respiratory capacity using oxygen electrodes

  • Assess mitochondrial membrane potential with fluorescent dyes (e.g., TMRM, JC-1)

  • Quantify ROS production using specific probes

• Energetic Status:

  • Determine ATP/ADP ratios

  • Measure energy charge using enzymatic assays

  • Monitor NAD⁺/NADH and NADP⁺/NADPH ratios

• Cellular Responses:

  • Analyze mitochondrial morphology and dynamics

  • Assess mitochondrial-nucleus retrograde signaling

  • Investigate cell death markers under severe stress

Integrated Experimental Design:

Data Integration and Systems Biology:

To fully understand the impact of stressors:

• Combine datasets using multivariate statistical approaches

• Apply metabolic flux analysis to quantify bioenergetic adjustments

• Develop predictive models of ATP synthase response to multiple stressors

• Compare responses across different Arabidopsis ecotypes to identify adaptive strategies

This comprehensive approach allows researchers to connect molecular changes in MI25 structure and function to physiological adaptations at the whole-plant level under environmental stress conditions.

What are common challenges in recombinant ATP synthase MI25 expression and how can they be overcome?

Recombinant expression of ATP synthase protein MI25 presents several challenges due to its nature as a hydrophobic membrane protein. Here are the most common issues and effective solutions:

Expression Challenges and Solutions:

• Low Expression Levels:

  Causes:

  • Codon bias in expression host

  • Protein toxicity to host cells

  • Poor mRNA stability

  Solutions:

  • Optimize codons for expression host

  • Use strains designed for toxic protein expression (C41/C43)

  • Reduce expression temperature to 16-20°C

  • Use tightly regulated promoters (e.g., araBAD instead of T7)

  • Include rare tRNA-expressing plasmids

• Protein Misfolding and Aggregation:

  Causes:

  • Insufficient membrane insertion machinery

  • Improper disulfide bond formation

  • Inadequate chaperone availability

  Solutions:

  • Express in hosts with enhanced membrane protein folding (C43, Lemo21)

  • Co-express with chaperones (GroEL/ES, DnaK/J)

  • Add membrane-mimetic compounds (glycerol 5-10%, specific lipids)

  • Use fusion partners that enhance solubility (MBP, SUMO)

• Protein Degradation:

  Causes:

  • Recognition by host proteases

  • Instability of partially folded intermediates

  Solutions:

  • Add protease inhibitors during purification

  • Use protease-deficient host strains

  • Optimize buffer conditions (pH, salt concentration)

  • Include stabilizing agents (glycerol, specific lipids)

Expression System-Specific Optimization:

Construct Design Optimization:

• Tag Selection and Placement:

  • N-terminal tags may interfere with membrane insertion

  • C-terminal tags are often preferred for membrane proteins

  • Consider dual tags for detection and purification

• Expression Vector Features:

  • Include pelB or other leader sequences for membrane targeting

  • Use vectors with adjustable induction levels

  • Consider using vectors with chaperone co-expression options

• Sequence Modifications:

  • Remove problematic sequences (internal ribosome binding sites)

  • Consider truncating non-essential regions

  • Introduce stabilizing mutations identified in homologous proteins

Validation and Quality Control:

Always verify expression through multiple detection methods:

• Western blotting with tag-specific and protein-specific antibodies

• Mass spectrometry confirmation of expressed protein

• Functional assays to confirm proper folding

By systematically addressing these challenges, researchers can significantly improve the expression of recombinant ATP synthase protein MI25 while maintaining its structural and functional integrity.

How can researchers troubleshoot issues in ATP synthase activity assays involving MI25?

ATP synthase activity assays can present various technical challenges when working with MI25-containing complexes. Effective troubleshooting requires systematic identification and resolution of issues:

Common Assay Issues and Solutions:

• Low or No Detectable Activity:

  Possible Causes:

  • Enzyme denaturation during purification

  • Detergent interference with enzyme function

  • Insufficient coupling between FO and F1 sectors

  • Improper buffer conditions

  Solutions:

  • Verify intact complex by BN-PAGE

  • Reduce detergent concentration to minimal working level

  • Test different detergents (DDM, digitonin, LMNG)

  • Optimize buffer components (pH 7.0-8.0, 5-10 mM Mg²⁺)

  • Add cardiolipin or other stabilizing lipids

• High Background Activity:

  Possible Causes:

  • Contaminating ATPases

  • Non-enzymatic ATP hydrolysis

  • Impurities in reagents

  Solutions:

  • Include specific inhibitors of other ATPases (e.g., ouabain for Na⁺/K⁺-ATPase)

  • Perform oligomycin sensitivity test (ATP synthase specific inhibitor)

  • Run parallel assays with heat-inactivated enzyme

  • Use higher purity ATP and other reagents

• Poor Reproducibility:

  Possible Causes:

  • Enzyme instability

  • Variable protein concentration measurement

  • Temperature fluctuations during assay

  Solutions:

  • Aliquot enzyme preparations to avoid freeze-thaw cycles

  • Use more accurate protein quantification methods (amino acid analysis)

  • Maintain strict temperature control during assays

  • Standardize all pipetting steps and incubation times

Assay-Specific Troubleshooting:

• ATP Hydrolysis Assays (Colorimetric):

  Issue	Solution
  Color development interference	Use detergent-compatible formulations
  Non-linear response	Ensure reaction is in linear range by testing multiple time points
  High blank values	Pre-incubate ATP solution to hydrolyze contaminating phosphate

• Proton Pumping Assays (Fluorescence-based):

  Issue	Solution
  Poor signal-to-noise ratio	Optimize protein:lipid ratio in proteoliposomes
  Rapid fluorescence decay	Check for proton leakage through damaged vesicles
  No response to ionophores	Verify successful protein reconstitution in liposomes

• ATP Synthesis Assays (Luciferase-based):

  Issue	Solution
  High background luminescence	Pre-treat reagents to remove ATP contamination
  Rapid signal decay	Use ATP regenerating system in the detection mixture
  Poor coupling to proton gradient	Optimize reconstitution conditions

Validation and Controls:

Always include these essential controls:

• Positive control: Commercial F1-ATPase or well-characterized ATP synthase

• Negative control: Heat-inactivated enzyme

• Inhibitor control: Oligomycin to distinguish ATP synthase activity

• Substrate control: Vary ATP concentration to determine kinetic parameters

Advanced Troubleshooting:

If basic troubleshooting fails, consider more sophisticated approaches:

• Structural analysis to check protein integrity (cryo-EM, CD spectroscopy)

• Mass spectrometry to verify complete subunit composition

• Crosslinking studies to confirm proper subunit interactions

• Single-molecule techniques to bypass ensemble averaging issues

By methodically addressing these potential issues, researchers can establish reliable activity assays for ATP synthase complexes containing MI25.

What strategies can overcome challenges in structural studies of ATP synthase complexes containing MI25?

Structural studies of ATP synthase complexes containing MI25 present unique challenges due to the large size, membrane-embedded nature, and conformational heterogeneity of the complex. Here are effective strategies to overcome these obstacles:

Sample Preparation Challenges:

• Protein Stability and Homogeneity:

  Challenges:

  • Dissociation of the complex during purification

  • Multiple conformational states

  • Detergent-induced structural changes

  Solutions:

  • Screen multiple detergents (DDM, digitonin, GDN, LMNG)

  • Use lipid nanodiscs or amphipols to maintain native environment

  • Add specific lipids (cardiolipin) known to stabilize ATP synthase

  • Employ GraFix technique (gradient fixation) for stabilization

  • Consider mild crosslinking to maintain subunit interactions

• Sample Concentration and Purity:

  Challenges:

  • Aggregation at high concentrations

  • Contaminants affecting structural analysis

  Solutions:

  • Use concentration devices with appropriate molecular weight cutoffs

  • Add stabilizers (glycerol, sucrose) during concentration

  • Implement additional purification steps (ion exchange, affinity chromatography)

  • Perform analytical ultracentrifugation to verify homogeneity

Cryo-EM Specific Strategies:

• Grid Preparation Optimization:

  Challenges:

  • Preferred orientation of particles

  • Particle aggregation at air-water interface

  • Poor ice quality

  Solutions:

  • Test different grid types (Quantifoil, C-flat, UltrAuFoil)

  • Apply thin carbon or graphene oxide support films

  • Use detergents like CHAPSO to modify air-water interface

  • Optimize blotting conditions (time, force, humidity)

  • Test Spotiton or Chameleon systems for more consistent vitrification

• Data Collection and Processing:

  Challenges:

  • Conformational heterogeneity

  • Low signal-to-noise ratio

  • Limited particle number

  Solutions:

  • Collect larger datasets (>5000 micrographs)

  • Use energy filters to improve contrast

  • Implement 3D classification to separate conformational states

  • Apply local refinement approaches for F1 and FO domains separately

  • Use particle subtraction to focus on regions of interest

Crystallography Approaches:

While challenging, X-ray crystallography can provide complementary information:

• Crystallization Strategies:

  • Use antibody fragments (Fabs) to create crystal contacts

  • Try lipidic cubic phase (LCP) crystallization for membrane regions

  • Focus on stable subcomplexes if whole complex proves recalcitrant

• Data Collection Optimization:

  • Utilize microfocus beamlines for small crystals

  • Consider serial crystallography approaches

  • Implement helical data collection for needle-like crystals

Hybrid Approaches:

Combining multiple structural methods can overcome limitations of individual techniques:

Case Study: Successful Strategy for Brine Shrimp ATP Synthase

As demonstrated in research on brine shrimp ATP synthase , a successful approach included:

• Solubilization with DDM at pH 7.0

• Purification by differential centrifugation and PEG precipitation

• Size exclusion chromatography for final purification

• Data collection with direct electron detectors

• 3D classification to separate rotational states

• Local refinement of F1 and FO domains separately, achieving resolutions of 2.6 Å and 3.9 Å respectively

By implementing these strategies, researchers overcame inherent challenges to reveal unprecedented structural details of ATP synthase.

What are the key unresolved questions about Arabidopsis thaliana ATP synthase protein MI25?

Despite significant advances in understanding ATP synthase structure and function, several critical questions about Arabidopsis thaliana ATP synthase protein MI25 remain unresolved:

Structural and Functional Uncertainties:

Physiological and Evolutionary Questions:

Technological and Methodological Challenges:

Priority Research Targets:

Addressing these unresolved questions will significantly advance our understanding of MI25's role in plant bioenergetics and potentially lead to applications in improving plant stress tolerance and agricultural productivity.

How might research on Arabidopsis thaliana ATP synthase protein MI25 contribute to improving plant stress tolerance?

Research on Arabidopsis thaliana ATP synthase protein MI25 has significant potential to enhance plant stress tolerance through multiple avenues:

Fundamental Insights with Applied Implications:

• Energy Efficiency Optimization:

  • Understanding how MI25 structure affects ATP synthase coupling efficiency

  • Identifying variants with improved performance under stress conditions

  • Engineering modifications that maintain ATP production during stress

• Stress-Responsive Regulation:

  • Elucidating how MI25 function is modulated during stress responses

  • Identifying regulatory mechanisms that could be enhanced

  • Developing methods to control ATP synthase activity during stress

• Cell Death Regulation:

  • Understanding MI25's contribution to mitochondrial permeability transition

  • Modulating controlled cell death responses during stress

  • Enhancing plant survival through optimized energy allocation

Translational Research Pathways:

• Genetic Engineering Approaches:

  • Introduction of stress-tolerant MI25 variants from extremophile plants

  • Fine-tuning expression levels under stress conditions

  • Coordinated modification of interacting proteins

• Screening and Breeding Applications:

  • Development of molecular markers based on MI25 sequence variations

  • Identification of naturally occurring beneficial alleles

  • Integration into marker-assisted breeding programs

• Chemical Biology Strategies:

  • Design of compounds that stabilize ATP synthase under stress

  • Development of molecules that modulate specific MI25 interactions

  • Creation of synthetic regulatory circuits controlling ATP synthase function

Specific Stress Applications:

Case Study: Theoretical Application to Drought Tolerance

A comprehensive approach to improving drought tolerance through MI25 research would involve:

• Comparative Analysis: Identify natural MI25 variants in drought-tolerant Arabidopsis ecotypes or related desert plants

• Functional Characterization: Determine how these variants affect:

  • ATP synthase efficiency under low water potential

  • Proton leak characteristics

  • Stability of the complex during dehydration

  • Interaction with stress-responsive proteins

• Engineering Strategy: Introduce beneficial modifications through:

  • Directed mutagenesis of specific residues

  • Domain swapping with drought-tolerant species

  • Adjustment of expression levels during drought progression

• Validation Pipeline: Test engineered plants for:

  • Maintenance of ATP levels during water limitation

  • Enhanced water use efficiency

  • Improved recovery after drought

  • Minimal yield penalties under normal conditions

Integration with Systems Biology:

For maximum impact, MI25 research should be integrated with:

• Global metabolic modeling to predict energetic outcomes

• Transcriptomic analysis to coordinate nuclear and organellar responses

• Phenomic approaches to connect molecular modifications to whole-plant outcomes

By pursuing these research directions, insights into MI25 function can contribute significantly to developing crops with enhanced resilience to environmental stressors, addressing critical needs in sustainable agriculture under changing climate conditions.

How can emerging technologies advance our understanding of ATP synthase protein MI25 structure and function?

Emerging technologies present unprecedented opportunities to deepen our understanding of ATP synthase protein MI25's structure and function:

Advanced Structural Biology Approaches:

• Cryo-Electron Tomography (Cryo-ET):

  • Visualize ATP synthase in its native cellular context

  • Study MI25's organization within the intact mitochondrial membrane

  • Examine interactions with other mitochondrial complexes

  • Achievable resolution now approaching 10 Å for in situ studies

• Time-Resolved Cryo-EM:

  • Capture transient conformational states during catalytic cycle

  • Visualize dynamic changes in MI25 during proton translocation

  • Combine with microfluidic devices for millisecond time resolution

  • Enable visualization of the complete rotational cycle

• Integrative Structural Biology:

  • Combine multiple data sources (cryo-EM, XL-MS, EPR, NMR)

  • Generate comprehensive structural models including flexible regions

  • Incorporate molecular dynamics simulations for dynamic understanding

  • Predict functional consequences of structural variations

Next-Generation Functional Analysis:

• Advanced Microscopy Techniques:

  • Super-resolution microscopy (PALM/STORM) to visualize individual complexes

  • FRET sensors to monitor conformational changes in vivo

  • Light-sheet microscopy for whole-organelle dynamics

  • Correlative light and electron microscopy (CLEM) to connect function and structure

• Single-Molecule Biophysics:

  • Optical tweezers to measure force generation during rotation

  • Magnetic tweezers to apply controlled torque to ATP synthase

  • High-speed AFM to visualize conformational dynamics in real-time

  • Zero-mode waveguides for single-molecule fluorescence at physiological concentrations

• Electrical Recording Techniques:

  • Solid-state nanopores to study channel properties

  • Advanced patch-clamp approaches for reconstituted ATP synthase

  • Planar lipid bilayer recordings with improved temporal resolution

  • Ion conductance microscopy for localized measurements

Genetic and Molecular Engineering Tools:

• Precision Genome Editing:

  • CRISPR-Cas9 modification of organellar genomes

  • Base editors for precise single-nucleotide modifications

  • Prime editing for targeted rewriting of MI25 sequence

  • DNA-free approaches using ribonucleoprotein complexes

• Synthetic Biology Approaches:

  • Minimal ATP synthase systems with defined components

  • Orthogonal translation systems for non-canonical amino acid incorporation

  • Engineered assembly pathways for modified complexes

  • Genetically encoded sensors reporting on ATP synthase function

• Optogenetic and Chemogenetic Control:

  • Light-controlled ATP synthase activity

  • Chemically inducible conformational changes

  • Spatiotemporal control of MI25 expression

  • Reversible inhibition systems

Computational and AI-Driven Approaches:

• Advanced Modeling Techniques:

  • Molecular dynamics simulations at extended timescales

  • Quantum mechanics/molecular mechanics (QM/MM) for catalytic mechanism

  • Markov state models of conformational transitions

  • Machine learning-assisted structure prediction

• Systems Biology Integration:

  • Multi-scale modeling from atoms to cellular networks

  • Constraint-based modeling of energetic consequences

  • Agent-based simulations of ATP synthase populations

  • Digital twin development for in silico experimentation

Implementation Roadmap for MI25 Research:

By strategically applying these emerging technologies, researchers can develop a comprehensive understanding of MI25's structure and function, from atomic-level mechanisms to cellular integration, ultimately enabling rational design of improved plant energy systems with enhanced stress tolerance.

What are the key considerations for researchers designing experiments involving Arabidopsis thaliana ATP synthase protein MI25?

Researchers designing experiments involving Arabidopsis thaliana ATP synthase protein MI25 should consider several critical factors to ensure robust, reproducible, and insightful results:

Experimental Design Fundamentals:

• Clear Research Questions and Hypotheses:

  • Formulate specific, testable hypotheses about MI25 function

  • Design experiments with appropriate controls that directly address the hypotheses

  • Consider both positive and negative outcomes in planning

• Appropriate Model Systems:

  • Whole plants for physiological relevance

  • Cell cultures for rapid screening and manipulation

  • Isolated mitochondria for direct bioenergetic assessment

  • Reconstituted systems for mechanistic studies

• Comprehensive Controls:

  • Genetic controls (wild-type, known mutants, complementation lines)

  • Technical controls (methodology validation, instrument calibration)

  • Environmental controls (consistent growth conditions, randomized designs)

  • Physiological controls (developmental stage matching, tissue selection)

Technical and Methodological Considerations:

• Protein Expression and Purification:

  • Select expression systems based on experimental needs

  • Maintain protein stability through appropriate buffer conditions

  • Verify protein integrity before functional studies

  • Consider native vs. recombinant approaches based on research questions

• Functional Assays:

  • Match assay sensitivity to expected effect size

  • Validate assays with known controls and standards

  • Perform preliminary experiments to establish optimal conditions

  • Consider multiple complementary assays to confirm findings

• Structural Studies:

  • Select appropriate techniques based on resolution needs

  • Consider native environment vs. purified systems

  • Plan for conformational heterogeneity

  • Integrate structural data with functional analyses

Biological Context Awareness:

• Developmental and Tissue Specificity:

  • Consider developmental timing of ATP synthase expression

  • Account for tissue-specific differences in energy demands

  • Match experimental conditions to relevant physiological states

  • Interpret findings in the context of plant growth stage

• Environmental Responsiveness:

  • Control or systematically vary environmental conditions

  • Consider diurnal patterns in energy metabolism

  • Account for stress history of experimental plants

  • Design realistic stress treatments that mimic natural conditions

• Genetic Background Effects:

  • Consider ecotype-specific variations

  • Account for modifier genes that may influence phenotypes

  • Generate multiple independent transgenic/mutant lines

  • Use complementation studies to confirm causality

Practical Research Implementation:

Ethical and Responsible Research Practices:

• Remember that robust experimental design is an ethical obligation

• Maintain comprehensive records of protocols and raw data

• Be transparent about limitations and failed attempts

• Consider environmental impact of research practices

By thoughtfully addressing these considerations, researchers can design experiments that yield meaningful insights into the structure, function, and physiological significance of ATP synthase protein MI25 in Arabidopsis thaliana.