Recombinant Agrobacterium vitis ATP synthase subunit alpha (atpA), partial

What is the role of ATP synthase subunit alpha in Agrobacterium vitis?

ATP synthase subunit alpha (atpA) in A. vitis forms part of the F1 catalytic domain of the bacterial ATP synthase complex. This complex is responsible for generating ATP by harnessing the energy from proton gradients across the cell membrane. The alpha subunit works in conjunction with the beta subunit to form the catalytic core where ATP synthesis occurs. Similar to other systems, such as chloroplast ATP synthase, the alpha subunit (AtpA) is one of the essential components responsible for enzymatic activity . For research purposes, understanding the structural and functional characteristics of atpA provides insights into A. vitis bioenergetics.

Why study partial recombinant atpA rather than the complete protein?

Studying partial recombinant atpA offers several methodological advantages:

• Expression efficiency: Partial proteins often express at higher levels with better solubility than full-length membrane-associated proteins

• Domain-specific analysis: Allows focused study on specific functional domains (e.g., nucleotide binding sites)

• Reduced complexity: Eliminates potentially problematic regions (hydrophobic segments, disordered regions)

• Crystallization potential: Improved probability of obtaining structural data through X-ray crystallography

• Epitope mapping: Facilitates identification of specific antigenic determinants

For researchers investigating specific aspects of atpA function, using a partial construct enables targeted analysis while avoiding technical challenges associated with full-length membrane protein expression.

What expression systems yield the highest functional protein for recombinant A. vitis atpA?

Several expression systems can be employed for recombinant A. vitis atpA production, each with specific advantages:

For optimal results with A. vitis atpA, the E. coli BL21(DE3) system using a pET vector with a C-terminal His-tag and expression at 18°C has proven effective for similar bacterial ATP synthase subunits. Addition of fusion partners such as MBP (maltose-binding protein) may further enhance solubility for partial atpA constructs.

What purification strategy yields the highest purity of recombinant A. vitis atpA?

A multi-step purification approach is recommended for obtaining high-purity recombinant A. vitis atpA:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged protein

  • Buffer recommendation: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, with gradient elution (20-250 mM imidazole)

• Intermediate purification:

  • Ion exchange chromatography based on the theoretical pI of atpA

  • Buffer recommendation: 20 mM Tris-HCl pH 8.0 with 0-500 mM NaCl gradient

• Final polishing:

  • Size exclusion chromatography to remove aggregates and obtain homogeneous protein

  • Buffer recommendation: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 1 mM DTT

This strategy typically yields >95% pure protein suitable for functional and structural studies. For studies requiring higher purity, additional chromatography steps such as hydroxyapatite chromatography may be incorporated.

How can expression and solubility issues with recombinant A. vitis atpA be resolved?

Recombinant bacterial proteins like A. vitis atpA often present expression and solubility challenges. Methodological solutions include:

• For low expression levels:

  • Optimize codon usage for expression host

  • Test different promoter strengths

  • Screen multiple expression vectors and fusion tags

  • Validate expression construct sequencing to ensure correct reading frame

• For protein insolubility:

  • Lower induction temperature (16-20°C)

  • Reduce inducer concentration (0.1 mM IPTG)

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

  • Use solubility-enhancing fusion partners (MBP, SUMO, TRX)

  • Include mild detergents (0.05% Triton X-100) during extraction

• For protein instability:

  • Add stabilizing agents (5-10% glycerol, 1 mM DTT)

  • Optimize buffer pH and ionic strength

  • Include protease inhibitors during purification

  • Test arginine and glutamic acid additions (50-100 mM)

These approaches have proven effective for other bacterial ATP synthase components and should be systematically tested for A. vitis atpA.

What assays can determine if recombinant A. vitis atpA retains native-like properties?

Multiple complementary approaches should be employed to verify that recombinant A. vitis atpA maintains native characteristics:

• Structural integrity assessment:

  • Circular dichroism spectroscopy to analyze secondary structure elements

  • Intrinsic fluorescence to probe tertiary structure and folding

  • Thermal shift assays to evaluate protein stability

  • Limited proteolysis to examine domain organization

• Functional assays:

  • ATP binding assays using TNP-ATP fluorescence

  • ATPase activity measurements via malachite green phosphate detection

  • Nucleotide binding kinetics via isothermal titration calorimetry

• Interaction studies:

  • Pull-down assays with other ATP synthase subunits

  • Surface plasmon resonance to quantify binding kinetics

  • Native PAGE to detect complex formation

Similar approaches have been successfully applied to characterize interactions between subunits of chloroplast ATP synthase, where the interaction between YL1 and AtpB subunits was established through complementary techniques .

How can site-directed mutagenesis be applied to study catalytic residues in A. vitis atpA?

Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in ATP synthase components:

• Target selection strategy:

  • Conserved residues identified through multi-species sequence alignments

  • Residues in predicted catalytic sites based on homology models

  • Positively charged residues potentially involved in phosphate binding

  • Residues at predicted subunit interfaces

• Mutagenesis experimental design:

  • Conservative mutations (e.g., Lys→Arg) to maintain charge but alter geometry

  • Non-conservative mutations (e.g., Lys→Ala) to eliminate functionality

  • Charge reversal mutations (e.g., Lys→Glu) to test electrostatic interactions

  • Systematic alanine scanning of targeted regions

• Functional impact assessment:

  • Measure changes in ATP binding affinity

  • Quantify effects on catalytic rates and efficiency

  • Determine alterations in protein stability

  • Analyze disruptions to protein-protein interactions

When interpreting mutagenesis results, it's essential to distinguish between direct effects on catalysis versus indirect effects on protein folding or complex assembly. Multiple complementary assays should be performed for each mutant.

How do buffer conditions affect recombinant A. vitis atpA stability and activity?

Buffer composition significantly impacts both stability and activity of ATP synthase components:

For activity assays, the buffer should mimic physiological conditions while maintaining protein stability. A starting buffer for A. vitis atpA characterization is 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5 mM MgCl₂, 1 mM DTT, and 10% glycerol, with systematic variation of components to identify optimal conditions.

How can the interaction between A. vitis atpA and other ATP synthase subunits be studied?

Multiple complementary approaches can reveal subunit interactions within the ATP synthase complex:

• In vitro binding assays:

  • Pull-down assays with recombinant subunits

  • Surface plasmon resonance for binding kinetics

  • Isothermal titration calorimetry for thermodynamic parameters

  • Microscale thermophoresis for interaction analysis

• Cross-linking approaches:

  • Chemical cross-linking followed by mass spectrometry (XL-MS)

  • Photo-affinity labeling to capture transient interactions

  • Site-specific incorporation of photo-cross-linkable amino acids

• Hybrid structural methods:

  • Cryo-electron microscopy of reconstituted complexes

  • Small-angle X-ray scattering (SAXS) of subunit combinations

  • Hydrogen-deuterium exchange mass spectrometry to map interfaces

• Cellular methods:

  • Bacterial two-hybrid systems

  • Fluorescence resonance energy transfer (FRET)

  • Co-immunoprecipitation from A. vitis extracts

Similar methods have been successfully applied to study interactions between YL1 and AtpB in chloroplast ATP synthase using yeast two-hybrid and bimolecular fluorescence complementation assays .

What structural prediction methods are most reliable for modeling A. vitis atpA?

For accurate structural modeling of A. vitis atpA, a hierarchical approach combining multiple methods is recommended:

• Template selection and evaluation:

  • Identify homologous structures from bacterial ATP synthases

  • Prioritize templates with highest sequence identity to A. vitis atpA

  • Evaluate template quality (resolution, R-factors, Ramachandran statistics)

  • Consider using multiple templates for different domains

• Modeling software selection strategy:

  • SWISS-MODEL for high sequence identity templates (>40%)

  • I-TASSER for more distant homologs

  • AlphaFold2 for highest accuracy in contemporary modeling

  • Rosetta for loop modeling and refinement

• Model validation protocol:

  • PROCHECK for stereochemical quality

  • VERIFY3D for sequence-structure compatibility

  • ProSA-web for knowledge-based energy assessment

  • MolProbity for all-atom contact analysis

• Refinement methodology:

  • Energy minimization using molecular mechanics force fields

  • Limited molecular dynamics simulations to relax strained regions

  • Side chain repacking in interface regions

  • Loop refinement for variable regions

When applying these methods to atpA, particular attention should be paid to the nucleotide binding pocket and regions involved in subunit interactions, as these are functionally critical and likely to be conserved.

How do recombinant A. vitis atpA catalytic properties compare with those from other bacterial species?

Understanding catalytic differences between ATP synthase alpha subunits from different bacterial species provides evolutionary and functional insights:

• Experimental design for comparative analysis:

  • Express recombinant atpA from multiple bacterial species using identical systems

  • Standardize purification protocols to ensure comparable protein quality

  • Employ consistent assay conditions across all protein variants

  • Include appropriate controls (e.g., catalytically inactive mutants)

• Kinetic parameters to measure:

  • ATP binding affinity (Kd) via isothermal titration calorimetry

  • Catalytic efficiency (kcat/Km) using coupled enzyme assays

  • pH optimum and profile across physiological range

  • Temperature dependence and activation energy

• Regulatory differences to examine:

  • Response to different nucleotides (ATP, GTP, CTP)

  • Inhibitor sensitivity profiles

  • Allosteric regulation mechanisms

  • Subunit interactions affecting catalysis

• Data analysis approach:

  • Statistical comparison of kinetic parameters

  • Principal component analysis of activity profiles

  • Structure-function correlation with molecular features

  • Phylogenetic analysis in relation to catalytic properties

These comparative analyses can reveal adaptations in A. vitis ATP synthase related to its specific ecological niche and lifestyle as a plant pathogen.

How can aggregation issues be resolved when working with recombinant A. vitis atpA?

Protein aggregation is a common challenge with ATP synthase components. Methodological solutions include:

• During expression:

  • Lower expression temperature (16-18°C)

  • Reduce induction strength (0.1 mM IPTG)

  • Co-express with molecular chaperones

  • Use specialized strains (e.g., ArcticExpress, SHuffle)

• During purification:

  • Maintain protein at concentrations below aggregation threshold

  • Include mild detergents (0.03% DDM or 0.05% CHAPS)

  • Add stabilizing agents (5-10% glycerol, 100-200 mM L-arginine)

  • Implement on-column refolding for inclusion bodies

• For storage and handling:

  • Optimize buffer composition (pH, salt concentration)

  • Store at moderate concentrations (1-2 mg/ml)

  • Add reducing agents to prevent disulfide-mediated aggregation

  • Consider flash-freezing in small aliquots with cryoprotectants

• Analysis of aggregation:

  • Dynamic light scattering to monitor size distribution

  • Size exclusion chromatography to quantify oligomeric states

  • Analytical ultracentrifugation for detailed aggregation analysis

  • ThT fluorescence to detect amyloid-like aggregation

Each approach should be systematically tested and optimized for A. vitis atpA based on empirical results.

How can contradictory results in ATP synthase activity assays be reconciled?

When facing inconsistent results in ATP synthase activity measurements:

• Systematic methodology validation:

  • Compare multiple assay methods (coupled enzymatic, colorimetric, radioisotopic)

  • Validate assay components with positive controls (commercial ATPases)

  • Determine assay detection limits and linear range

  • Calculate signal-to-noise ratio and optimization

• Protein quality assessment:

  • Verify homogeneity by size exclusion chromatography

  • Assess stability over time at assay temperature

  • Confirm protein concentration using multiple methods

  • Check for inhibitory contaminants from purification

• Experimental condition analysis:

  • Test activity across pH range (pH 6.0-9.0)

  • Vary ionic strength systematically (50-500 mM)

  • Titrate divalent cation concentration (1-20 mM Mg²⁺)

  • Examine temperature dependence (20-40°C)

• Data analysis approaches:

  • Implement appropriate statistical methods for significance testing

  • Use global fitting for complex kinetic models

  • Apply outlier detection algorithms

  • Consider alternative mechanistic models

When reporting results, transparently document all methods, conditions, and variations to allow reproducibility and proper interpretation by the scientific community.

What strategies can overcome poor yield of recombinant A. vitis atpA?

Low protein yield can be addressed through systematic optimization:

• Expression vector optimization:

  • Test multiple promoter strengths (T7, tac, araBAD)

  • Optimize codon usage for expression host

  • Adjust 5' untranslated region for optimal translation initiation

  • Screen different fusion tags (His6, GST, MBP, SUMO)

• Host strain selection:

  • Compare standard strains (BL21, Rosetta) with specialized strains

  • Test strains with extra chaperones (Arctic Express, OverExpress)

  • Consider strains with altered redox environment (SHuffle, Origami)

  • Evaluate toxicity through growth curve analysis

• Culture condition optimization:

  • Screen media formulations (LB, TB, auto-induction)

  • Test different induction points (early, mid, late log phase)

  • Optimize inducer concentration through systematic titration

  • Evaluate co-additives (sorbitol, betaine, ethanol, metal ions)

• Protein extraction enhancement:

  • Compare mechanical (sonication, homogenization) and chemical lysis

  • Test specialized extraction buffers with detergents

  • Implement on-column refolding for inclusion bodies

  • Optimize cell density at harvest

For challenging proteins like A. vitis atpA, combining multiple approaches is often necessary to achieve acceptable yields. Establishing a Design of Experiments (DoE) approach can efficiently identify optimal combinations of these variables.

How does A. vitis atpA structure-function relationship compare to plant chloroplast ATP synthase?

Comparing bacterial and plant chloroplast ATP synthases provides evolutionary insights:

• Structural similarities:

  • Both contain alpha/beta hexameric arrangements in the F1 catalytic domain

  • Core catalytic mechanisms conserved across domains of life

  • Similar nucleotide binding sites and catalytic residues

• Key differences:

  • Chloroplast ATP synthase contains plant-specific regulatory subunits

  • Chloroplast ATP synthase is regulated by light-dependent processes

  • A. vitis ATP synthase likely evolved for bacterial physiological needs

• Functional implications:

  • Bacterial ATP synthases primarily function in ATP production during respiration

  • Chloroplast ATP synthases coordinate with photosynthetic electron transport

  • Different regulatory mechanisms reflect distinct energy management strategies

Research on chloroplast ATP synthase has demonstrated that specific factors like YL1 are required for efficient biogenesis and assembly . Such specialized assembly factors might have bacterial counterparts in A. vitis that could be identified through comparative genomics.

What insights from T-DNA transfer mechanisms in A. vitis can inform ATP synthase research?

The well-characterized T-DNA transfer process in A. vitis provides contextual understanding for ATP synthase research:

• Physiological connections:

  • Energy requirements during infection and gene transfer processes

  • ATP synthase activity changes during host interaction

  • Potential metabolic adaptations during plant colonization

• Research methodology crossover:

  • Genetic manipulation techniques established for virulence studies

  • Host interaction models that influence metabolic regulation

  • Strain characterization methods applicable to bioenergetic studies

Studies characterizing A. vitis strains have revealed different host range capabilities , suggesting potential metabolic adaptations that might involve energy production systems. Researchers studying A. vitis ATP synthase should consider how these virulence mechanisms interact with cellular bioenergetics during host infection.

How do post-translational modifications affect A. vitis atpA function compared to other bacterial systems?

Post-translational modifications (PTMs) can significantly impact ATP synthase function:

• Common ATP synthase PTMs in bacteria:

  • Phosphorylation affecting catalytic activity and regulation

  • Acetylation influencing protein stability and interactions

  • Oxidative modifications during stress responses

• Analytical approaches for PTM identification:

  • LC-MS/MS analysis with enrichment strategies

  • Phospho-specific antibodies for targeted detection

  • Chemical labeling approaches for specific modifications

  • Targeted proteomic methods for modification quantification

• Functional consequences to investigate:

  • Activity regulation through reversible modifications

  • Complex assembly and stability effects

  • Stress response and environmental adaptation mechanisms

• Experimental design for PTM studies:

  • Compare PTM profiles under different growth conditions

  • Generate site-directed mutants at modified residues

  • Develop in vitro modification systems to test direct effects

  • Compare modification patterns across related bacterial species

This research area remains largely unexplored for A. vitis and represents an opportunity to discover novel regulatory mechanisms for bacterial ATP synthases.

How can cryo-electron microscopy advance understanding of A. vitis ATP synthase structure?

Cryo-EM offers transformative potential for ATP synthase structural studies:

• Advantages for ATP synthase research:

  • Visualization of complete ATP synthase complexes in near-native states

  • No crystallization requirement, overcoming a major hurdle for membrane proteins

  • Ability to capture different conformational states during catalytic cycle

  • Potential for subnanometer resolution of the entire complex

• Sample preparation considerations:

  • Detergent selection for membrane extraction (DDM, LMNG)

  • Reconstitution into nanodiscs to mimic native membrane environment

  • Vitrification conditions optimization to prevent preferred orientation

  • Concentration balancing to avoid aggregation while maintaining visibility

• Data collection and processing strategy:

  • Collection of large datasets (>5000 micrographs) to capture rare conformations

  • Use of energy filters and phase plates to enhance contrast

  • Particle picking optimization for heterogeneous samples

  • 3D classification to separate distinct conformational states

• Integration with complementary methods:

  • Molecular dynamics simulations to model dynamic regions

  • Crosslinking mass spectrometry to validate subunit interactions

  • Functional assays to correlate structure with activity states

This approach would enable visualization of how A. vitis ATP synthase subunits, including atpA, assemble into a functional complex.

How does the excision frequency of T-DNA in A. vitis relate to ATP synthase function during infection?

Connecting T-DNA excision with ATP synthase function opens new research directions:

• Potential metabolic connections:

  • Energy requirements during T-DNA processing and transfer

  • ATP-dependent steps in the infection process

  • Metabolic changes during different stages of plant interaction

• Experimental approaches:

  • Monitor ATP synthase activity during T-DNA excision

  • Compare energy metabolism in strains with different excision frequencies

  • Analyze effects of ATP synthase inhibitors on T-DNA transfer

  • Measure ATP levels in bacteria during plant interaction

Research on A. vitis has shown that T-DNA excision can be stimulated by grapevine tissues but not by acetosyringone (AS), differing from A. tumefaciens where the opposite effect is observed . This suggests unique signaling pathways in A. vitis that may interact with energy metabolism regulation.

How can systems biology approaches integrate A. vitis atpA function into cellular networks?

Systems-level analysis provides comprehensive understanding of ATP synthase in cellular context:

• Multi-omics integration strategy:

  • Combine transcriptomics, proteomics, and metabolomics data

  • Correlate ATP synthase expression with metabolic states

  • Identify regulatory networks controlling bioenergetics

  • Map protein-protein interaction networks centered on ATP synthase

• Metabolic modeling approaches:

  • Construct genome-scale metabolic models for A. vitis

  • Perform flux balance analysis to predict energy requirements

  • Simulate the effects of environmental changes on ATP demand

  • Model metabolic adaptation during host interaction

• Network analysis methods:

  • Identify hub proteins connecting energy metabolism to other processes

  • Perform pathway enrichment analysis for associated functions

  • Construct gene regulatory networks controlling ATP synthase expression

  • Compare networks across different growth conditions

• Experimental validation approaches:

  • Gene knockout studies of predicted regulators

  • Metabolic flux analysis using isotope tracers

  • Targeted proteomics to quantify pathway components

  • ChIP-seq to identify transcription factor binding sites

This systems approach would place A. vitis ATP synthase in the broader context of bacterial physiology and host-pathogen interactions, potentially revealing novel therapeutic targets.