Recombinant Rhodopirellula baltica ATP synthase subunit alpha 2 (atpA2), partial

What is the structure and function of ATP synthase in Rhodopirellula baltica?

ATP synthase in R. baltica likely follows the general structure of F-type ATP synthases with two main sectors: the membrane-embedded Fo sector and the catalytic F1 sector that protrudes into the cytoplasm. Based on comparative genomic analysis with other bacteria like Rhodobacter capsulatus, we can infer that in R. baltica, genes encoding the F1 sector are likely organized in an operon similar to the atpHAGDC operon . The F1 sector typically contains five subunits (α, β, γ, δ, and ε) with the composition α3β3γδε.

The ATP synthase functions by harnessing the proton gradient across the membrane to drive the synthesis of ATP from ADP and inorganic phosphate. The alpha subunit (encoded by atpA) contains nucleotide binding sites that are primarily regulatory rather than catalytic and works in conjunction with the beta subunit that contains the catalytic sites.

The presence of an atpA2 gene in R. baltica's genome suggests this organism has an alternative or additional alpha subunit, potentially expressed under specific environmental conditions or having specialized functions in its marine habitat.

How does the atpA2 subunit differ from atpA1 in R. baltica?

While the search results don't provide direct comparative information about R. baltica atpA1 and atpA2, typical differences in organisms with multiple ATP synthase alpha subunits include:

• Sequence homology: atpA2 likely shares significant sequence identity with atpA1, particularly in functional domains involved in nucleotide binding and interactions with other subunits.

• Expression conditions: Similar to other marine bacteria with multiple functional gene variants, atpA2 might be expressed under specific environmental conditions (pH, temperature, salt concentration) different from those triggering atpA1 expression.

• Functional adaptations: The atpA2 subunit might confer different properties to the ATP synthase complex, such as altered ATP synthesis rates, different substrate affinities, or modified regulatory responses.

• Evolutionary significance: The presence of multiple atpA genes likely represents gene duplication events that allowed R. baltica to adapt to the diverse and changing conditions of marine ecosystems.

Genomic analysis of Rhodopirellula species like strain P2 has revealed significant adaptations for marine environments, including specialized metabolic pathways . This suggests atpA2 may play a role in R. baltica's environmental adaptation strategies.

What expression systems are optimal for recombinant R. baltica atpA2 production?

The optimal expression system for recombinant R. baltica atpA2 depends on research objectives, with several methodological considerations:

• E. coli expression system:

  • Most commonly used due to rapid growth and established protocols

  • Optimal for initial characterization studies

  • Considerations: Codon optimization may be necessary due to different codon usage

  • Recommended strains: BL21(DE3) for general expression; C41(DE3) or C43(DE3) for potentially toxic membrane-associated proteins

  • Similar to protocols used for recombinant protein production in other studies

• Yeast expression systems (S. cerevisiae or P. pastoris):

  • Better for proteins requiring eukaryotic post-translational modifications

  • Slower growth but potentially better folding of complex proteins

  • Particularly useful if the recombinant protein affects E. coli viability, as observed with some ATP synthase components

• Cell-free expression systems:

  • Useful for rapid screening or when the protein is toxic to cells

  • Allows for incorporation of modified amino acids

  • Lower yields but faster turnaround time

Methodology for optimizing expression:

• Test multiple constructs with different tags (His, GST, MBP)

• Optimize induction conditions (temperature, IPTG concentration, induction time)

• Screen for solubility and activity in small-scale tests before scaling up

• Confirm protein identity by mass spectrometry

What purification methods work best for recombinant R. baltica atpA2?

Purification of recombinant R. baltica atpA2 typically follows a multi-step process designed to isolate the protein with high purity while maintaining its native conformation and activity:

• Affinity Chromatography:

  • If expressed with a His-tag: Ni-NTA purification as described for other recombinant proteins

  • Protocol: Equilibrate Ni-NTA resin with buffer (typically 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole); apply clarified lysate; wash extensively; elute with imidazole gradient (50-250 mM)

  • Alternative tags: GST-tag with glutathione resin or MBP-tag with amylose resin can improve solubility

• Ion Exchange Chromatography:

  • Based on the predicted isoelectric point of atpA2

  • Typically using Q-Sepharose (anion exchange) if the protein has a pI below 7

  • Buffer conditions: 20 mM Tris-HCl pH 8.0 with gradient elution using NaCl (0-500 mM)

• Size Exclusion Chromatography:

  • Final polishing step to separate monomeric protein from aggregates and other contaminants

  • Column selection based on expected molecular weight of atpA2 (typically Superdex 200)

  • Similar to procedures described for other recombinant proteins

• Quality Control:

  • SDS-PAGE to assess purity (>95% for most applications)

  • Western blot with anti-His antibodies or specific antibodies against atpA2

  • Mass spectrometry to confirm identity

  • Activity assays to confirm functionality

For particularly challenging purifications, additional specialized methods might be considered:

• Hydroxyapatite chromatography

• Hydrophobic interaction chromatography

• Tag removal using specific proteases (TEV, thrombin) if the tag affects function

How stable is recombinant R. baltica atpA2 under various storage conditions?

The stability of recombinant R. baltica atpA2 under different storage conditions must be determined empirically. Here's a methodological approach to assess and maximize stability:

• Short-term storage options:

  • 4°C: Typically stable for 1-2 weeks in appropriate buffer

  • Buffer optimization: Test stability in different buffers (HEPES, Tris, phosphate) with varying pH (7.0-8.0)

  • Stabilizing additives: Glycerol (10-20%), reducing agents (1-5 mM DTT or 1-2 mM β-mercaptoethanol), and salt concentration (50-300 mM NaCl)

• Long-term storage options:

  • -20°C: Addition of 50% glycerol typically prevents freezing damage

  • -80°C: Flash-freezing in liquid nitrogen and storage at -80°C, with or without cryoprotectants

  • Lyophilization: For very long-term storage, though activity recovery can be variable

• Methodology for stability assessment:

  • Activity assays at defined time points (0, 1 day, 1 week, 1 month, 3 months)

  • SDS-PAGE to monitor degradation

  • Dynamic light scattering to assess aggregation

  • Thermal shift assays to determine thermal stability under different conditions

Based on experience with other ATP synthase subunits, the protein is likely to be most stable when stored at -80°C after flash freezing in a buffer containing 20-25 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, and 1 mM DTT.

What are the critical factors affecting enzymatic activity of recombinant R. baltica atpA2?

The enzymatic activity of recombinant R. baltica atpA2 is influenced by multiple factors that should be carefully controlled and optimized in experimental designs:

• Structural integrity and proper folding:

  • Secondary structure analysis using circular dichroism spectroscopy

  • Thermal stability assessment using differential scanning fluorimetry

  • Native PAGE to confirm oligomeric state if applicable

• Buffer composition effects:

  • pH optimization (typically pH 6.5-8.5 for ATP synthase components)

  • Ionic strength (50-300 mM salt concentration)

  • Divalent cation requirements (Mg²⁺, Ca²⁺, Mn²⁺ at 1-10 mM)

  • Presence of specific lipids if the protein normally interacts with membranes

• Substrate considerations:

  • ATP/ADP concentration (typically 0.1-5 mM)

  • Testing both ATP synthesis and hydrolysis directions

  • Phosphate concentration effects

  • Nucleotide specificity (ATP vs. GTP vs. other nucleotides)

• Experimental design for activity measurements:

  • Coupled enzyme assays (e.g., with pyruvate kinase and lactate dehydrogenase)

  • Direct ATPase activity measurement using malachite green phosphate assay

  • Determination of kinetic parameters (Km, Vmax, kcat)

• Potential inhibitors and activators:

  • Sensitivity to known ATP synthase inhibitors (oligomycin, DCCD, aurovertin)

  • Product inhibition analysis

  • Allosteric regulation investigation

It's important to note that atpA2 alone may not show ATP synthase activity, as it typically functions as part of the larger F₁ complex. Researchers might need to reconstitute it with other subunits (particularly β, γ, δ, and ε) to observe physiologically relevant activity, similar to the approach used for the ATP synthase from R. capsulatus .

How can post-translational modifications of recombinant R. baltica atpA2 be analyzed?

Post-translational modifications (PTMs) of recombinant R. baltica atpA2 could significantly impact its function and interactions. Here's a comprehensive methodological approach to analyze potential PTMs:

• Mass Spectrometry-Based Approaches:

  • Sample preparation: In-gel or in-solution digestion with multiple proteases (trypsin, chymotrypsin) to ensure comprehensive coverage

  • LC-MS/MS analysis: High-resolution instruments like Orbitrap or Q-TOF for accurate mass determination

  • Data analysis workflow:
    a) Database searching using tools like Mascot, SEQUEST, or MaxQuant
    b) Variable modification settings for common PTMs (phosphorylation, acetylation, methylation)
    c) Manual verification of spectra for identified modified peptides

• Specific PTM Detection Methods:

  • Phosphorylation:
    a) ProQ Diamond phosphoprotein staining of SDS-PAGE gels
    b) Western blotting with anti-phospho antibodies (Ser, Thr, Tyr)
    c) Phos-tag SDS-PAGE for mobility shift detection

  • Glycosylation:
    a) Periodic acid-Schiff (PAS) staining
    b) Lectin binding assays with different specificities
    c) Mass shift analysis after treatment with specific glycosidases

  • Other modifications:
    a) Western blotting with anti-acetyllysine antibodies
    b) Chemical labeling approaches for specific modifications

• Functional Impact Assessment:

  • Site-directed mutagenesis of modified residues

  • Activity assays comparing wild-type and mutant proteins

  • Structural analysis of modification sites using available structural data

When analyzing PTMs, it's important to consider that recombinant expression in E. coli may not reproduce the same modification pattern as in the native R. baltica due to differences in the PTM machinery. For more authentic modification patterns, expression in systems more closely related to the native organism might be considered.

What strategies can resolve conflicting kinetic data for recombinant R. baltica atpA2?

Conflicting kinetic data for recombinant R. baltica atpA2 can arise from various sources including experimental conditions, protein preparation differences, or assay variations. Here's a methodological approach to resolve such conflicts:

• Standardization of Protein Preparation:

  • Develop a detailed SOP (Standard Operating Procedure) for expression and purification

  • Implement consistent quality control metrics:
    a) Purity assessment (>95% by SDS-PAGE)
    b) Activity benchmarks against known standards
    c) Protein concentration determination using multiple methods (Bradford, BCA, A280)

  • Aliquot single batches for comparative studies to eliminate batch-to-batch variation

• Systematic Analysis of Experimental Variables:

  • Create a matrix of experimental conditions to test:

    Variable	Range to Test	Increments	Critical Considerations
    pH	6.0-9.0	0.5 units	Marine bacteria often have pH adaptations
    Temperature	10-50°C	5°C steps	R. baltica is mesophilic
    [NaCl]	0-500 mM	50 mM steps	Marine environment adaptation
    [Mg²⁺]	0-20 mM	2 mM steps	Essential for ATP binding
    Substrate [ATP/ADP]	0.01-10 mM	Half-log steps	Concentration-dependent kinetics

  • Use statistical design of experiments (DOE) to identify significant factors and interactions

  • Determine optimal conditions where activity is most reproducible

• Assay Method Comparison:

  • Compare direct and coupled assay systems:
    a) Malachite green phosphate detection
    b) Coupled enzyme assays with spectrophotometric detection
    c) Luciferase-based ATP detection
    d) ³²P-ATP radioactive assays

  • Assess each method for sensitivity, dynamic range, and potential interfering factors

  • Develop correction factors if necessary for comparing data between methods

• Mathematical Modeling of Kinetic Behavior:

  • Beyond basic Michaelis-Menten kinetics, explore:
    a) Allosteric models (Hill equation, MWC model)
    b) Product inhibition models
    c) Two-substrate kinetic models for ATP synthase activity

  • Use global fitting approaches across multiple datasets

Example reconciliation table for conflicting Km values:

*Adjusted to standard conditions of pH 7.5, 25°C, 5 mM Mg²⁺

This systematic approach helps distinguish whether discrepancies represent true biological variability, experimental artifacts, or methodological differences, leading to more robust and reproducible kinetic parameters.

How can site-directed mutagenesis be optimized for studying R. baltica atpA2 function?

Site-directed mutagenesis is a powerful approach for investigating structure-function relationships in R. baltica atpA2. Here's a methodological framework for optimizing this technique:

• Strategic Target Selection:

  • Rational design based on:
    a) Sequence alignment with well-characterized ATP synthase alpha subunits
    b) Structural homology modeling using related crystal structures
    c) Evolutionary conservation analysis across bacterial species, particularly marine bacteria

  • Key regions to target:
    a) Nucleotide binding sites
    b) Subunit interface regions
    c) Catalytic residues
    d) Regulatory sites
    e) Unique residues specific to atpA2 vs. atpA1

• Mutagenesis Method Optimization:

  • QuikChange site-directed mutagenesis:
    a) Primer design optimization (length 25-45 bp, Tm ≥78°C, GC clamp)
    b) Template considerations (methylated plasmid DNA, low concentration)
    c) PCR optimization (extension time, DMSO addition for GC-rich regions)

  • Alternatives for difficult templates:
    a) Megaprimer method
    b) Gibson Assembly
    c) Golden Gate Assembly

  • Similar approaches have been used successfully for creating mutations in other bacterial genes

• Expression System Considerations:

  • Codon optimization for expression host

  • Selection of appropriate tags that don't interfere with the region being studied

  • Consideration of compensatory mutations if primary mutations affect stability

• Validation Strategy:

  • Sequencing to confirm mutations (entire gene, not just mutation site)

  • Expression level comparison between mutants and wild-type

  • Folding assessment using thermal shift assays or circular dichroism

  • Basic ATPase activity screening before detailed characterization

• Functional Analysis Framework:

  • Systematic categorization of mutants:

    Mutation Type	Expected Effect	Primary Assays	Secondary Assays
    Active site	Altered catalysis	Enzyme kinetics	Nucleotide binding
    Interface	Assembly defects	Size exclusion	Subunit interaction
    Regulatory	Response changes	Inhibitor studies	Allosteric response
    Stability	Folding issues	Thermal stability	Limited proteolysis

Example mutational analysis data presentation:

This systematic approach allows for comprehensive characterization of structure-function relationships in R. baltica atpA2 and can reveal unique properties compared to other bacterial ATP synthase alpha subunits.

How can recombinant R. baltica atpA2 be used to study bacterial adaptation to marine environments?

Recombinant R. baltica atpA2 offers unique opportunities to investigate bacterial adaptation to marine ecosystems. The presence of multiple ATP synthase alpha subunits in R. baltica likely represents an adaptive strategy for energy metabolism in varying marine conditions.

• Comparative Functional Assays:

  • Measure enzymatic activity across environmental parameters:
    a) Salt concentration gradients (0-1000 mM NaCl)
    b) Temperature ranges (4-40°C)
    c) pH variation (pH 6.0-9.0)
    d) Pressure effects (1-100 atm)

  • Compare atpA1 vs. atpA2 performance under these conditions

  • Correlate findings with R. baltica's natural habitat conditions

• Evolutionary Analysis:

  • Phylogenetic comparisons with ATP synthase subunits from:
    a) Other marine bacteria
    b) Freshwater bacteria
    c) Terrestrial bacteria

  • Identification of marine-specific adaptations in protein sequence

  • Molecular clock analysis to date the gene duplication event

• Expression Pattern Analysis:

  • Develop quantitative PCR assays to measure relative expression of atpA1 vs. atpA2

  • Determine environmental triggers for differential expression

  • Correlate with proteomic data from R. baltica under various growth conditions

• Recombination Experiments:

  • Create chimeric proteins combining domains from atpA1 and atpA2

  • Test functional properties of chimeras under various conditions

  • Identify specific regions responsible for environmental adaptations

This research could provide insights into how R. baltica and related Planctomycetota have adapted their energy metabolism to thrive in marine environments, similar to studies that have revealed other adaptations in Rhodopirellula species like the ability to degrade complex marine polysaccharides .