Recombinant Oligotropha carboxidovorans ATP synthase subunit delta (atpH)

What is the structural role of the delta subunit in Oligotropha carboxidovorans ATP synthase?

Basic Research Question

The delta subunit (atpH) of ATP synthase in Oligotropha carboxidovorans serves as a critical connector between the F₁ catalytic domain and the F₀ membrane domain. It forms part of the peripheral stalk that prevents rotation of the α₃β₃ hexamer during ATP synthesis. Structurally, the delta subunit typically consists of an N-terminal domain that binds to the F₁ sector and a C-terminal domain that interacts with the F₀ sector.

Methodologically, researchers should investigate the structural properties through:

• X-ray crystallography to determine atomic resolution structure

• Cryo-electron microscopy for visualization within the complete ATP synthase complex

• Comparative structural analysis with well-characterized delta subunits from model organisms like E. coli

• Hydrogen-deuterium exchange mass spectrometry to identify regions with differential solvent accessibility

How does the amino acid sequence of O. carboxidovorans ATP synthase delta subunit compare to other bacterial species?

Basic Research Question

The O. carboxidovorans ATP synthase delta subunit shares moderate sequence conservation with other bacterial homologs, with highest conservation in regions involved in subunit interactions. While specific sequence alignment data for this particular subunit is limited in current literature, researchers typically observe 30-60% sequence identity between delta subunits across different bacterial species.

For robust sequence analysis, researchers should:

• Retrieve the amino acid sequence from protein databases (UniProt, NCBI)

• Perform multiple sequence alignment with CLUSTAL Omega or MUSCLE

• Generate a phylogenetic tree using Maximum Likelihood or Bayesian methods

• Calculate conservation scores for each position using ConSurf or similar tools

• Map conservation patterns onto available structural models to identify functional motifs

What experimental approaches can determine the oligomeric state of recombinant O. carboxidovorans ATP synthase delta subunit?

Advanced Research Question

Determining the oligomeric state is essential for understanding functional properties. For the O. carboxidovorans ATP synthase delta subunit, researchers should employ multiple complementary techniques:

• Size Exclusion Chromatography (SEC): Separates proteins based on hydrodynamic radius and provides initial insights into oligomeric state.

• Analytical Ultracentrifugation: Both sedimentation velocity and equilibrium experiments can accurately determine molecular weight and homogeneity. This approach has been successfully used for characterizing related enzyme complexes .

• Multi-Angle Light Scattering (MALS): When coupled with SEC, MALS provides absolute molecular weight determination independent of shape.

• Chemical Crosslinking followed by Mass Spectrometry: Captures transient interactions between subunits and identifies specific interaction interfaces.

• Native Mass Spectrometry: Determines precise oligomeric distributions in native-like conditions.

What are the optimal expression systems for producing functional recombinant O. carboxidovorans ATP synthase delta subunit?

Basic Research Question

The choice of expression system significantly impacts yield, solubility, and functionality. For O. carboxidovorans ATP synthase delta subunit, researchers should consider:

• Bacterial Expression Systems:

  • E. coli BL21(DE3) or its derivatives are preferred for expressing bacterial proteins

  • Cold-induction strategies (15-18°C) improve solubility

  • Codon-optimized constructs enhance expression by 2-5 fold

  • Fusion partners (MBP, SUMO, TrxA) increase solubility

• Cell-Free Expression Systems:

  • Allow rapid screening of expression conditions

  • Useful for proteins that might be toxic to host cells

  • Provide immediate access for biophysical characterization

Methodologically, researchers should optimize expression by testing:

• Various promoter strengths (T7, tac, araBAD)

• Induction conditions (IPTG concentration: 0.1-1.0 mM; temperature: 15-37°C)

• Multiple fusion tags (His-tag, GST, MBP) at N- and C-termini

• Different cell lysis methods to preserve structure and function

Recent commercial production of related recombinant Oligotropha carboxidovorans ATP synthase subunits suggests bacterial expression systems are suitable for the delta subunit as well .

What purification strategy yields the highest purity and activity of recombinant O. carboxidovorans ATP synthase delta subunit?

Advanced Research Question

Purifying recombinant O. carboxidovorans ATP synthase delta subunit to homogeneity while maintaining native conformation requires a multi-step approach:

• Initial Capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Glutathione-S-transferase (GST) affinity chromatography for GST-fusion proteins

  • Ammonium sulfate fractionation as an alternative initial step

• Intermediate Purification:

  • Ion exchange chromatography (IEX) based on the theoretical pI of the delta subunit

  • Hydrophobic interaction chromatography (HIC)

• Polishing:

  • Size exclusion chromatography (SEC) for final purity and assessment of oligomeric state

  • Removal of affinity tags if necessary for functional studies

Based on purification protocols described for related enzymes, a typical purification table might appear as follows :

Methodologically, researchers should optimize each purification step by testing different buffer compositions (pH 6.5-8.5, salt concentration 50-500 mM), stabilizing additives (glycerol, reducing agents), and storage conditions to maintain long-term stability.

How can researchers assess the folding status and structural integrity of purified recombinant O. carboxidovorans ATP synthase delta subunit?

Advanced Research Question

Ensuring proper folding and structural integrity is crucial for functional studies. Researchers should employ multiple biophysical techniques:

• Circular Dichroism (CD) Spectroscopy:

  • Far-UV CD (190-250 nm) estimates secondary structure content

  • Near-UV CD (250-350 nm) probes tertiary structure

  • Thermal denaturation studies assess stability

• Fluorescence Spectroscopy:

  • Intrinsic tryptophan fluorescence monitors tertiary structure

  • ANS binding detects exposed hydrophobic patches

  • Thermal and chemical denaturation curves provide stability parameters

• Limited Proteolysis:

  • Comparison of digestion patterns between purified protein and denatured controls

  • Mass spectrometry analysis of fragments identifies flexible regions

• Differential Scanning Calorimetry (DSC):

  • Determines thermal stability and domain organization

Similar approaches have been successfully applied to related enzymes, such as circular dichroism spectroscopy to assess structural differences between wild-type and mutant forms of related enzymes .

What are the recommended methods for assessing the functional role of the delta subunit in ATP synthase activity?

Basic Research Question

While the delta subunit alone doesn't possess catalytic activity, it plays critical roles in complex assembly and function:

• ATP Synthase Reconstitution Assays:

  • Reconstitute ATP synthase with and without the delta subunit

  • Measure ATP synthesis/hydrolysis rates using:

    • Malachite green assay for phosphate detection

    • Luciferin-luciferase assay for ATP quantification

    • Coupled enzyme assays linking ATP hydrolysis to NADH oxidation

• Proton Pumping Assays:

  • Reconstitute ATP synthase into liposomes

  • Measure pH changes using pH-sensitive fluorescent dyes

  • Compare proton pumping efficiency with and without the delta subunit

• Rotational Catalysis Measurements:

  • Single-molecule FRET to monitor conformational changes

  • Fluorescence microscopy with attached beads to visualize rotation

Methodologically, researchers should:

• Ensure high-quality protein preparations

• Test activity across a range of pH values (6.0-9.0) and temperatures (25-55°C)

• Determine kinetic parameters (Km, Vmax, kcat)

• Assess the effects of known inhibitors

Similar enzyme activity assays have been successfully developed for related enzyme systems with careful optimization of reaction conditions .

How can researchers investigate the interaction between the delta subunit and other components of the ATP synthase complex?

Advanced Research Question

Understanding protein-protein interactions within the ATP synthase complex requires multiple complementary approaches:

• Surface Plasmon Resonance (SPR):

  • Determine binding kinetics and affinity constants

  • Compare wild-type and mutant proteins to identify critical interaction sites

  • Typical experimental design includes immobilizing the delta subunit and flowing other subunits as analytes

• Isothermal Titration Calorimetry (ITC):

  • Measure thermodynamic parameters (ΔH, ΔS, ΔG)

  • Determine binding stoichiometry

  • Assess the contribution of entropy vs. enthalpy to binding

• Chemical Cross-Linking coupled with Mass Spectrometry:

  • Map interaction interfaces at the residue level

  • Identify distance constraints between subunits

  • Use zero-length and variable-length crosslinkers to probe different spatial relationships

• Förster Resonance Energy Transfer (FRET):

  • Monitor interactions in real-time

  • Measure distances between specific sites using site-directed labeling

  • Detect conformational changes during catalysis

Similar approaches have been used to characterize protein-protein interactions in related enzyme systems, providing insights into complex assembly and subunit cooperation .

What spectroscopic methods are most informative for characterizing recombinant O. carboxidovorans ATP synthase delta subunit?

Advanced Research Question

Spectroscopic methods provide valuable insights into protein structure, dynamics, and function:

• UV-Visible Spectroscopy:

  • Monitor protein concentration and purity

  • Detect chromophoric cofactors if present

  • Follow conformational changes that alter chromophore environments

• Circular Dichroism (CD) Spectroscopy:

  • Quantify secondary structure elements (α-helices, β-sheets)

  • Monitor conformational changes upon ligand binding or pH changes

  • Assess thermal stability through melting curves

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Obtain residue-specific structural information

  • Map protein-protein interaction surfaces

  • Study conformational dynamics at different timescales

• Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Study local environments through site-directed spin labeling

  • Measure distances between labeled sites

  • Detect conformational changes during function

UV-visible spectroscopy and EPR spectroscopy have been successfully applied to characterize related enzymes, as detailed in research on similar enzyme systems . For example, EPR spectroscopy has been used to identify specific radical species formed during catalysis in related oxidoreductase enzymes.

How can site-directed mutagenesis be used to investigate the functional roles of conserved residues in O. carboxidovorans ATP synthase delta subunit?

Advanced Research Question

Site-directed mutagenesis enables systematic investigation of structure-function relationships:

• Rational Design of Mutations:

  • Target conserved residues identified through sequence alignment

  • Focus on charged residues at potential interaction interfaces

  • Create mutations that alter specific properties (charge, size, hydrophobicity)

• Comprehensive Mutation Strategy:

  • Alanine-scanning mutagenesis to systematically probe residue contributions

  • Conservative substitutions to maintain side chain properties

  • Non-conservative substitutions to disrupt specific interactions

• Functional Analysis of Mutants:

  • Binding affinity measurements with partner subunits

  • Assembly efficiency into the ATP synthase complex

  • Effects on ATP synthesis/hydrolysis activities

  • Structural stability and conformational dynamics

Similar mutagenesis approaches revealed how single residue mutations significantly impact enzyme function in related systems. For example, the E331A mutation in ACS decreased enzyme activity by approximately 25-fold, demonstrating the critical role of this residue in catalysis .

How can hydrogen-deuterium exchange mass spectrometry (HDX-MS) inform our understanding of O. carboxidovorans ATP synthase delta subunit dynamics?

Advanced Research Question

HDX-MS has emerged as a powerful technique for studying protein dynamics, conformational changes, and protein-protein interactions:

• Mapping Structural Dynamics:

  • Identify regions with high flexibility versus stable core regions

  • Detect conformational changes upon binding to other subunits

  • Reveal allosteric effects of nucleotide binding

• Characterizing Interaction Interfaces:

  • Monitor changes in deuterium uptake upon complex formation

  • Pinpoint specific segments involved in protein-protein interactions

  • Compare binding interfaces across different states (ATP vs. ADP-bound)

• Experimental Approach:

  • Incubate protein in D2O buffer for varying time periods (10 sec to 24 hours)

  • Quench exchange with cold acidic conditions

  • Perform pepsin digestion and LC-MS analysis

  • Compare deuterium incorporation between different states

• Data Analysis:

  • Generate deuterium uptake curves for each peptide

  • Calculate protection factors

  • Create heat maps of exchange rates mapped onto structural models

While HDX-MS was not specifically mentioned in the search results, the detailed structural and mechanistic studies described for related enzymes suggest that this technique would provide valuable insights into ATP synthase component dynamics .

What are the most effective approaches for studying the role of O. carboxidovorans ATP synthase delta subunit in energy coupling?

Advanced Research Question

Understanding how the delta subunit contributes to energy coupling requires sophisticated biophysical and biochemical approaches:

• Single-Molecule Techniques:

  • Fluorescence microscopy to visualize rotation of the γ subunit

  • Magnetic bead rotation assays to measure torque generation

  • Optical tweezers to apply controlled forces and measure mechanical responses

• Reconstitution Studies:

  • Liposome reconstitution with wild-type or mutant delta subunits

  • Measurement of proton gradient formation and ATP synthesis coupling

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

• Cryo-EM Analysis:

  • Structure determination in different catalytic states

  • Visualization of conformational changes during the catalytic cycle

  • Mapping of the delta subunit position during rotation

• Molecular Dynamics Simulations:

  • Model conformational changes during the rotary mechanism

  • Predict the effect of mutations on energy transmission

  • Simulate interactions between subunits during catalysis

Similar multidisciplinary approaches combining structural methods, biochemical assays, and computational techniques have been successfully applied to study related enzyme systems, as evidenced by the detailed mechanistic understanding achieved for enzymes like oxalate oxidoreductase .

How does the function of ATP synthase delta subunit in O. carboxidovorans compare to that in other bacterial species?

Basic Research Question

Comparative analysis provides insights into conserved functions and species-specific adaptations:

• Functional Conservation:

  • The core role in connecting F₁ and F₀ complexes is preserved across species

  • The function in preventing futile rotation of the α₃β₃ hexamer is universal

  • Involvement in coupling proton translocation to ATP synthesis is maintained

• Species-Specific Adaptations:

  • Variations in thermostability reflecting the organism's growth environment

  • Differences in pH optimum corresponding to the organism's ecological niche

  • Adaptations to specific energy metabolism (carbon monoxide utilization in O. carboxidovorans)

Methodologically, researchers should perform comparative biochemical characterization using identical experimental conditions across different bacterial ATP synthases, focusing on:

• Stability under different temperature and pH conditions

• ATP synthesis/hydrolysis kinetics

• Proton pumping efficiency

• Subunit interaction strengths

What insights can be gained from studying the co-evolution of ATP synthase subunits in O. carboxidovorans?

Advanced Research Question

Co-evolutionary analysis explores how interacting proteins or protein domains evolve in concert:

• Computational Coevolution Analysis:

  • Direct coupling analysis (DCA) to identify co-evolving residue pairs

  • Statistical coupling analysis (SCA) to detect co-evolving networks

  • Mutual information (MI) calculations to quantify correlated mutations

• Correlation with Structural Data:

  • Map co-evolving residue pairs onto structural models

  • Identify interaction networks spanning multiple subunits

  • Predict functional importance of co-evolving regions

• Experimental Validation:

  • Mutagenesis of co-evolving residue pairs to test functional importance

  • Rescue experiments using compensatory mutations

  • Structural analysis of mutant proteins to verify predicted interactions

This approach has proven valuable for understanding the evolutionary constraints on multi-subunit enzyme complexes, complementing the structural and functional studies described for related enzyme systems .

How can structural biology approaches contribute to our understanding of the ATP synthase delta subunit in the context of the complete enzyme complex?

Advanced Research Question

Modern structural biology techniques provide unprecedented insights into the architecture and dynamics of large macromolecular assemblies:

• Cryo-Electron Microscopy (cryo-EM):

  • Determine structures of the complete ATP synthase complex

  • Visualize the delta subunit in its native context

  • Capture different conformational states during the catalytic cycle

  • Typical resolution ranges from 2.5-4.0 Å for well-behaved complexes

• Integrative Structural Biology:

  • Combine cryo-EM with X-ray crystallography of individual domains

  • Incorporate distance constraints from crosslinking mass spectrometry

  • Use small-angle X-ray scattering (SAXS) to validate solution structures

  • Integrate dynamics information from HDX-MS and NMR

• Time-Resolved Structural Studies:

  • Use time-resolved cryo-EM to capture short-lived intermediates

  • Employ temperature-jump techniques to synchronize conformational changes

  • Correlate structural snapshots with functional states

• In Situ Structural Biology:

  • Cryo-electron tomography of ATP synthase in native membranes

  • Correlative light and electron microscopy to study localization and structure

  • Sub-tomogram averaging to improve resolution of in situ structures

While these approaches were not specifically described in the search results for O. carboxidovorans ATP synthase, the detailed structural investigations of related enzyme complexes highlight the power of structural biology for understanding complex molecular machines .