Recombinant Jannaschia sp. ATP synthase subunit b' (atpG)

How does the structure of Jannaschia sp. ATP synthase subunit b' compare to homologous proteins in other bacterial species?

Sequence alignment studies reveal that Jannaschia sp. ATP synthase subunit b' shares significant structural homology with similar subunits from other α-proteobacteria, including:

Notably, bacterial subunit b (b' or b2 isoform) from α-proteobacteria like Jannaschia sp. has evolutionary significance as it shares ancestry with mitochondrial subunit 8 (A6L) . The N-terminal region of Jannaschia sp. ATP synthase subunit b' contains a membrane-spanning domain, while the C-terminal region extends into the cytoplasm and interacts with other structural components of the ATP synthase complex.

This evolutionary relationship provides important insights into the endosymbiotic theory, as mitochondrial subunit 8 appears to be a vestige of bacterial subunit b that remained encoded in mitochondrial DNA .

What are the recommended methods for expression and purification of recombinant Jannaschia sp. ATP synthase subunit b'?

Based on established protocols for similar ATP synthase subunits, the following methodological approach is recommended:

Expression System Selection:

• E. coli expression systems are typically most effective for bacterial membrane proteins

• BL21(DE3) or Rosetta(DE3) strains are preferred for membrane protein expression

• Expression vectors containing T7 promoters provide controlled, high-level expression

Expression Protocol:

• Transform expression plasmid containing codon-optimized atpG gene into competent cells

• Culture in LB-glucose medium (1.0% tryptone, 0.5% yeast extract, 0.4% glucose, 0.5% NaCl) with appropriate antibiotics

• Grow at 37°C to an optical density of 0.6-0.7

• Induce expression with IPTG (1.0 mM) for 3-4 hours or at lower temperature (18°C) overnight

• Harvest cells by centrifugation at ~6000 × g for 20 minutes

Purification Strategy:

• Resuspend cell pellet in lysis buffer (typically 20 mM Tris-HCl pH 8.0 with protease inhibitors)

• Disrupt cells via sonication or French press

• Isolate membrane fraction through differential centrifugation

• Solubilize membrane proteins using mild detergents (DDM, LDAO, or C12E8)

• Purify using affinity chromatography (if tagged) followed by size exclusion chromatography

• Store in Tris-based buffer with 50% glycerol at -20°C or -80°C for extended storage

Critical Note: Repeated freezing and thawing should be avoided. Working aliquots should be stored at 4°C for up to one week .

What are the optimal storage conditions for maintaining stability of purified Jannaschia sp. ATP synthase subunit b'?

The recombinant protein requires specific storage conditions to maintain structural integrity and function:

• Short-term storage (≤1 week): Store working aliquots at 4°C in Tris-based buffer

• Medium-term storage: Store at -20°C in buffer containing 50% glycerol as a cryoprotectant

• Long-term storage: Store at -80°C in buffer containing 50% glycerol

To prevent protein degradation:

• Add protease inhibitors to the storage buffer

• Divide the purified protein into single-use aliquots to avoid repeated freeze-thaw cycles

• Ensure rapid freezing to minimize ice crystal formation

• Consider adding reducing agents (e.g., DTT or β-mercaptoethanol) if the protein contains cysteine residues

As explicitly noted in the product information, repeated freezing and thawing is strongly discouraged as it can lead to protein denaturation and loss of structural integrity .

How can researchers design experiments to investigate the role of Jannaschia sp. ATP synthase subunit b' in the functional assembly of the complete ATP synthase complex?

To investigate the role of subunit b' in ATP synthase assembly, researchers should consider the following experimental design approaches:

In vitro Reconstitution Studies:

• Express and purify individual ATP synthase components, including subunit b' (wild-type and mutant variants)

• Perform systematic reconstitution of the ATP synthase complex with and without subunit b'

• Assess complex stability using Blue Native PAGE or analytical ultracentrifugation

• Evaluate enzyme activity through ATP hydrolysis and synthesis assays

• Analyze interaction interfaces using chemical cross-linking coupled with mass spectrometry

Mutagenesis Approaches:

• Generate targeted mutations in conserved regions of subunit b'

• Assess the impact on complex assembly using co-immunoprecipitation or pull-down assays

• Evaluate structural implications through circular dichroism or thermal stability assays

• Examine functional consequences through ATP synthesis/hydrolysis activity measurements

Cryo-EM Structural Analysis:
Recent studies on yeast and bacterial ATP synthases using cryo-EM have provided valuable structural insights . Similar approaches for Jannaschia sp. ATP synthase could:

• Visualize the structural organization of subunit b' within the complete complex

• Identify specific interaction interfaces with other subunits

• Reveal conformational changes associated with enzyme function

This integrative approach would elucidate the structural and functional role of subunit b' in maintaining the connection between the catalytic F₁ domain and the membrane-embedded F₀ domain, which is critical for the rotary mechanism of ATP synthesis.

What biophysical techniques are most effective for characterizing protein-protein interactions involving Jannaschia sp. ATP synthase subunit b'?

Several advanced biophysical techniques can be employed to characterize the protein-protein interactions of subunit b':

Microscale Thermophoresis (MST):

• Measures biomolecular interactions based on thermophoresis changes

• Requires minimal sample amounts (typically in the μM range)

• Can detect interactions with components of varying sizes

• Suitable for membrane protein interactions when combined with appropriate detergents

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

• Maps protein interaction surfaces by monitoring differential deuterium uptake

• Identifies regions protected from exchange upon complex formation

• Particularly valuable for analyzing dynamic interactions in large complexes

• Can provide information on conformational changes upon binding

Surface Plasmon Resonance (SPR):

• Allows real-time monitoring of binding kinetics

• Determines association (k₁) and dissociation (k₋₁) rate constants

• Calculates equilibrium dissociation constants (Kᴅ)

• Requires immobilization of one binding partner

Isothermal Titration Calorimetry (ITC):

• Measures thermodynamic parameters of binding (ΔH, ΔS, ΔG)

• Provides stoichiometry information

• Does not require labeling or immobilization

• Useful for characterizing interactions in solution

Cross-linking Mass Spectrometry (XL-MS):

• Captures transient interactions through covalent cross-links

• Identifies specific residues involved in interactions

• Provides distance constraints for structural modeling

• Compatible with membrane protein complexes when using appropriate cross-linkers

These techniques should be applied in combination to generate comprehensive interaction profiles for subunit b' with other components of the ATP synthase complex.

How does the amino acid sequence of Jannaschia sp. ATP synthase subunit b' inform its structural role in the ATP synthase complex?

The amino acid sequence of Jannaschia sp. ATP synthase subunit b' provides critical insights into its structural and functional roles:

Sequence Analysis and Domain Organization:

The 193 amino acid sequence of Jannaschia sp. ATP synthase subunit b' (UniProt: Q28UC6) can be divided into functional domains:

• N-terminal membrane-anchoring domain (residues 1-35):

  • Contains hydrophobic residues forming a transmembrane helix

  • The segment "IFWLVLTLLAIYFVLTK" likely spans the membrane

  • Anchors the protein to the bacterial membrane

• Central flexible region (residues 36-90):

  • Contains charged and polar residues

  • Likely forms a flexible linker between membrane and peripheral domains

  • Critical for accommodating conformational changes during catalysis

• C-terminal peripheral stalk domain (residues 91-193):

  • Rich in charged and polar residues

  • Forms coiled-coil structures with other subunits

  • Responsible for interactions with the F₁ sector

Structure-Function Relationship:

The sequence suggests that subunit b' adopts an extended conformation, with its N-terminus embedded in the membrane and its C-terminus extending into the cytoplasm to interact with the F₁ catalytic domain. This arrangement is consistent with its role in providing a static connection between the F₁ and F₀ domains, resisting the torque generated during rotary catalysis.

Comparative sequence analysis with other bacterial b subunits reveals conserved features that maintain this structural role across species, despite sequence divergence . This conservation underscores the fundamental importance of subunit b' in the ATP synthase architecture.

What are the most significant challenges and solutions in studying the biochemical properties of recombinant Jannaschia sp. ATP synthase subunit b'?

Challenges and Methodological Solutions:

Advanced Solution - Chimeric Protein Approach:
For particularly challenging aspects of subunit b' biochemistry, researchers can create chimeric proteins where problematic regions are replaced with better-characterized homologous segments from related species. This approach maintains the essential functional domains while improving biochemical properties for in vitro studies.

The integration of these methodological solutions enables comprehensive biochemical characterization despite the inherent challenges associated with membrane protein components of large molecular complexes.

How can researchers evaluate the impact of post-translational modifications on Jannaschia sp. ATP synthase subunit b' function?

Methodological Approach for PTM Analysis:

• Identification of Potential PTMs:

  • Perform high-resolution mass spectrometry (LC-MS/MS) on purified native subunit b'

  • Use multiple proteases (trypsin, chymotrypsin, Glu-C) to ensure complete sequence coverage

  • Apply enrichment strategies for specific PTMs (e.g., TiO₂ for phosphopeptides)

  • Search for common bacterial PTMs: phosphorylation, acetylation, methylation, and glycosylation

• Validation of Identified PTMs:

  • Generate site-specific antibodies against modified peptides

  • Use targeted mass spectrometry (MRM/PRM) to quantify modification stoichiometry

  • Apply site-directed mutagenesis to create PTM-mimicking mutants (e.g., S→D for phosphorylation)

• Functional Impact Assessment:

  • Compare enzymatic activities of wild-type and PTM-mimicking mutants

  • Analyze ATP synthesis/hydrolysis rates in reconstituted systems

  • Evaluate complex assembly efficiency through BN-PAGE or analytical ultracentrifugation

  • Assess structural changes via circular dichroism or HDX-MS

• Physiological Relevance Investigation:

  • Identify conditions that alter PTM patterns (environmental stress, growth phase)

  • Analyze PTM dynamics during bacterial adaptation to different energy states

  • Compare PTM patterns across related bacterial species to identify conserved regulatory mechanisms

This comprehensive approach provides insights into how PTMs might regulate ATP synthase function in Jannaschia sp., potentially revealing novel regulatory mechanisms in bacterial energy metabolism.

What comparative genomic approaches can elucidate the evolutionary history of the atpG gene in Jannaschia sp. and related marine bacteria?

Methodological Framework for Evolutionary Analysis:

• Sequence Collection and Alignment:

  • Extract atpG gene sequences from complete genomes of Roseobacter clade members

  • Include sequences from diverse bacterial phyla as outgroups

  • Perform multiple sequence alignment using MAFFT or T-Coffee algorithms

  • Refine alignments manually to ensure accurate homology assessment

• Phylogenetic Analysis:

  • Construct maximum likelihood trees using RAxML or IQ-TREE

  • Apply appropriate substitution models (LG+G+F, WAG+I+G)

  • Perform bootstrap analysis (1000 replicates) to assess node support

  • Compare gene trees with species trees to identify potential horizontal gene transfer events

• Synteny Analysis:

  • Examine gene order conservation around atpG across species

  • Identify genomic rearrangements affecting ATP synthase operons

  • Map operon structures across diverse bacterial lineages

• Selection Pressure Analysis:

  • Calculate dN/dS ratios to identify signatures of selection

  • Apply site-specific models to detect positively selected residues

  • Correlate selection patterns with functional domains

• Structural Constraint Mapping:

  • Map evolutionary conservation onto structural models

  • Identify structurally constrained regions versus variable segments

  • Correlate evolutionary rates with functional importance

This comparative genomic approach reveals how atpG has evolved within the Roseobacter clade, which includes Jannaschia sp. and other marine bacteria. The analysis provides insights into the adaptation of ATP synthase to marine environments and the evolutionary constraints on this essential energy-producing complex.

In particular, this approach can illuminate the evolutionary relationship between bacterial subunit b' and mitochondrial subunit 8, which appears to be a vestige of bacterial subunit b that remained encoded in mitochondrial DNA .

What are the optimal conditions for analyzing protein-lipid interactions involving Jannaschia sp. ATP synthase subunit b'?

Methodological Framework for Protein-Lipid Interaction Analysis:

• Lipid Binding Assessment:

  Solid-state NMR spectroscopy provides the most comprehensive data on protein-lipid interactions in membrane environments. The following approach is recommended:

  • Reconstitute purified subunit b' into liposomes with defined lipid composition

  • Apply 2D and 3D solid-state NMR experiments to map lipid-protein contact points

  • Use paramagnetic relaxation enhancement (PRE) with spin-labeled lipids to identify specific interaction sites

  • Complement with native mass spectrometry to identify tightly bound lipids that co-purify with the protein

• Lipid Specificity Analysis:

  Technique	Application	Data Output
  Liposome flotation assays	Measures binding to specific lipid compositions	Binding affinity to different lipid mixtures
  Microscale thermophoresis	Detects interactions with labeled lipids	Quantitative binding parameters (Kd)
  Laurdan fluorescence spectroscopy	Measures effects on lipid packing	Changes in membrane organization
  Differential scanning calorimetry	Analyzes effects on lipid phase transitions	Thermodynamic parameters of lipid perturbation

• Functional Impact Analysis:

  To determine how lipid interactions affect function:

  • Reconstitute ATP synthase complexes in proteoliposomes with varying lipid compositions

  • Measure ATP synthesis/hydrolysis rates as a function of lipid environment

  • Assess proton translocation efficiency in different membrane contexts

  • Monitor protein stability and complex assembly in different lipid environments

These methods collectively provide insights into how the lipid environment modulates the structure and function of Jannaschia sp. ATP synthase subunit b', which is particularly relevant given its membrane-anchoring role in the ATP synthase complex.

How can researchers accurately quantify the stoichiometry and oligomeric state of Jannaschia sp. ATP synthase subunit b' in the native complex?

Methodological Approaches for Stoichiometry Determination:

• Native Mass Spectrometry:

  • Apply specialized membrane protein MS techniques with gentle ionization

  • Use nano-electrospray ionization with detergent-containing buffers

  • Carefully analyze peak distributions to determine subunit stoichiometry

  • Compare results with known stoichiometries of homologous ATP synthases

• Quantitative Crosslinking:

  • Apply isotopically labeled crosslinkers to intact ATP synthase complexes

  • Analyze crosslinked products by SDS-PAGE and mass spectrometry

  • Quantify relative abundances of intra- and inter-subunit crosslinks

  • Use modeling to determine compatible stoichiometric arrangements

• Single-Molecule Fluorescence Techniques:

  • Label subunit b' with fluorescent tags at specific positions

  • Apply single-molecule photobleaching step analysis

  • Count bleaching steps to determine number of subunits

  • Use Förster resonance energy transfer (FRET) to map spatial arrangements

• Analytical Ultracentrifugation:

  • Perform sedimentation velocity experiments on purified complexes

  • Analyze data using continuous size distribution models

  • Determine molecular weight of intact complexes

  • Compare with theoretical weights of different stoichiometric models

• Cryo-Electron Microscopy:

  • Obtain high-resolution structures of intact ATP synthase complexes

  • Apply single particle analysis to determine subunit arrangement

  • Use 3D classification to identify potential stoichiometric variations

  • Integrate with crosslinking data for validation

Based on structural data from related bacterial ATP synthases, it is anticipated that Jannaschia sp. ATP synthase would contain either one or two copies of subunit b', with the latter arrangement being more common in bacterial systems . Accurate determination of this stoichiometry is crucial for understanding the structural architecture and assembly mechanism of the complex.

What approaches can be used to investigate the role of Jannaschia sp. ATP synthase subunit b' in energy metabolism within its native marine bacterial context?

Integrative Research Framework:

• Genetic Manipulation Strategies:

  • Develop gene knockout/knockdown systems for Jannaschia sp.

  • Create conditional expression systems to control atpG levels

  • Generate site-directed mutants affecting key functional domains

  • Complement with wild-type or mutant variants to verify phenotypes

• Physiological Characterization:

  • Assess growth rates under different energy sources (photoheterotrophic vs. chemoheterotrophic)

  • Measure membrane potential using potential-sensitive dyes

  • Determine ATP/ADP ratios under various growth conditions

  • Analyze respiratory chain activity with specific inhibitors

• In vivo ATP Synthesis Measurements:

  • Apply luciferase-based ATP biosensors for real-time monitoring

  • Use 31P-NMR to measure ATP synthesis rates in intact cells

  • Determine P/O ratios (ATP synthesized per oxygen consumed)

  • Compare wild-type with atpG mutants to assess functional impact

• Ecological Context Experiments:

  • Simulate marine environmental conditions (temperature, salinity, nutrient limitation)

  • Conduct competition assays between wild-type and atpG mutants

  • Analyze gene expression profiles under different ecological stresses

  • Study impact on survival during nutrient limitation or oxidative stress

• Structural Biology in Native Context:

  • Apply in-cell NMR to study structural properties in intact cells

  • Use in situ cryo-electron tomography to visualize ATP synthase in native membranes

  • Employ chemical crosslinking in intact cells followed by MS analysis

  • Compare with in vitro reconstituted systems to identify context-dependent features

This comprehensive approach would provide insights into how Jannaschia sp. ATP synthase subunit b' contributes to energy metabolism in these marine photoheterotrophic bacteria, potentially revealing adaptations specific to their ecological niche.

What are the recommended protocols for generating and characterizing site-directed mutants of Jannaschia sp. ATP synthase subunit b'?

Comprehensive Mutagenesis Workflow:

• Rational Mutation Design:

  • Analyze sequence conservation across homologous proteins

  • Identify functionally important residues based on structural models

  • Design mutations targeting:

    • Membrane-spanning regions

    • Interface residues involved in subunit interactions

    • Charged residues that may participate in proton translocation

    • Regions implicated in assembly or stability

• Mutant Characterization:

  Analysis	Purpose	Expected Outcome
  Thermal stability assay	Assess structural integrity	ΔTm values compared to wild-type
  Circular dichroism	Evaluate secondary structure	Changes in α-helical or β-sheet content
  Membrane integration analysis	Verify proper membrane insertion	Accessibility to proteases or chemical labeling
  ATP synthase assembly assay	Assess incorporation into complex	Co-immunoprecipitation or BN-PAGE analysis
  Functional reconstitution	Measure impact on ATP synthesis	Changes in enzyme kinetics or coupling efficiency

• Advanced Mutant Analysis:

  • Apply hydrogen-deuterium exchange MS to map conformational changes

  • Use cross-linking MS to identify altered interaction networks

  • Perform molecular dynamics simulations to predict structural impacts

  • Create double or triple mutants to test functional hypotheses derived from single mutants

This systematic approach enables comprehensive functional dissection of Jannaschia sp. ATP synthase subunit b', providing insights into its role in ATP synthase assembly, stability, and catalytic function.

How can researchers effectively compare the properties of Jannaschia sp. ATP synthase subunit b' with its homologs from other bacterial species to understand functional adaptations?

Comparative Biochemistry Framework:

• Parallel Expression and Purification:

  • Express homologous subunit b' proteins from diverse bacterial sources:

    • Other Roseobacter clade members

    • Model organisms (E. coli, R. sphaeroides)

    • Extremophiles (thermophiles, halophiles)

  • Use identical expression systems and purification protocols

  • Standardize buffer conditions for direct comparison

• Structural Comparison:

  • Analyze secondary structure content via circular dichroism

  • Compare thermostability profiles using differential scanning calorimetry

  • Assess oligomerization tendencies through analytical ultracentrifugation

  • Determine solution structures using small-angle X-ray scattering

• Environmental Adaptation Analysis:

  Environmental Parameter	Experimental Approach	Insight Gained
  Temperature dependence	Activity assays at 5-45°C	Thermal adaptation of marine bacteria
  Salt tolerance	Stability in 0-1M NaCl	Halotolerance mechanisms
  pH sensitivity	Structure/function at pH 5-9	Proton handling adaptations
  Pressure effects	High-pressure biochemistry	Deep-sea adaptations

• Evolutionary Context Analysis:

  • Correlate biochemical properties with phylogenetic relationships

  • Analyze selection pressures on specific domains or residues

  • Identify convergent evolution patterns across ecological niches

  • Map biochemical adaptations to environmental conditions