Recombinant Acidovorax sp. ATP synthase subunit c (atpE)

What is the function of ATP synthase subunit c in Acidovorax species?

ATP synthase subunit c, encoded by the atpE gene, forms the critical oligomeric ring structure (C-ring) within the membrane-embedded F₀ sector of ATP synthase. In Acidovorax species, as in other bacteria, this subunit functions as an essential component of the proton-conducting pathway. The C-ring contains the ion-binding site necessary for proton translocation across the membrane, which drives the rotational catalysis that generates ATP . Each subunit c has a hairpin structure comprising two membrane-spanning α-helices connected by a hydrophilic loop at the cytoplasmic side of the membrane, with the essential ion carrier (glutamate or aspartate residue) located in the middle of the C-terminal α-helix . This structure enables the C-ring to rotate during proton translocation, coupling the electrochemical gradient to ATP synthesis in the F₁ sector of the complex.

What methods are recommended for PCR amplification of the Acidovorax sp. atpE gene?

For PCR amplification of the Acidovorax sp. atpE gene, researchers should consider a protocol similar to those used for mycobacterial ATP synthase genes but with primers specifically designed for Acidovorax sequences. DNA can be amplified using degenerate primers targeting conserved regions across bacterial atpE sequences, followed by more specific amplification with Acidovorax-specific primers . A typical approach includes:

• Initial DNA extraction using a standard bacterial genomic DNA extraction kit

• PCR amplification with degenerate primers (e.g., atpE-F: 5'-GARTTYATHGCNTGYTTYGG-3' and atpE-R: 5'-CCNARYCCRAANARRTCNCC-3')

• Verification of PCR products by agarose gel electrophoresis (1.5%)

• Purification of PCR products

• Cloning into a suitable vector such as pMOSBlue or other blunt-ended cloning systems

• Confirmation by sequencing

The PCR reaction can be optimized using a gradient of annealing temperatures (50-60°C) to find the optimal conditions for Acidovorax species. DNA polymerases with proofreading capability are recommended for high-fidelity amplification .

How does the structure of Acidovorax sp. ATP synthase subunit c compare to other bacterial species?

The structure of Acidovorax sp. ATP synthase subunit c likely shares common features with other bacterial c subunits while maintaining species-specific variations. While no crystal structure is available specifically for Acidovorax sp. C-ring, comparative analysis can be performed by homology modeling using known bacterial structures.

Based on comparative studies of other bacterial ATP synthases, we can infer that Acidovorax sp. subunit c likely forms a ring structure with 10-15 subunits, similar to other bacterial species which range from 8-15 subunits depending on the organism . Key structural features include:

• Two membrane-spanning α-helices connected by a hydrophilic loop

• A conserved ion-binding glutamate or aspartate residue at the middle of the C-terminal helix

• A proton-binding site located between adjacent c subunits in the assembled C-ring

The amino acid composition of subunit c varies significantly across species, but the residues involved in ion binding and ring formation are generally conserved. The Acidovorax sp. subunit c would likely maintain these critical functional residues while potentially showing differences in other regions that might affect drug binding or interactions with other ATP synthase components .

What are the optimal expression systems and purification strategies for obtaining functional recombinant Acidovorax sp. ATP synthase subunit c?

For efficient expression and purification of functional recombinant Acidovorax sp. ATP synthase subunit c, researchers should consider a multi-step strategy that accounts for the hydrophobic nature of this membrane protein. The following methodology is recommended:

Expression System Selection:

• E. coli C41(DE3) or C43(DE3) strains - These strains are engineered for membrane protein expression and show reduced toxicity effects

• pET vector system with a C-terminal His6-tag - Provides controlled expression and facilitates purification

• Low-temperature induction (16-18°C) - Slows expression rate, improving proper folding

Optimized Expression Protocol:

• Transform expression plasmid into selected E. coli strain

• Grow culture to OD600 of 0.6-0.8 at 37°C

• Reduce temperature to 18°C and induce with 0.1-0.5 mM IPTG

• Continue expression for 16-20 hours

Purification Strategy:

• Cell lysis by sonication or French press in buffer containing 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol

• Membrane fraction isolation by differential centrifugation

• Membrane solubilization using 1% n-dodecyl-β-D-maltoside (DDM) or 1% digitonin

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin

• Size exclusion chromatography for final purification and oligomeric state assessment

For functional studies, the purified subunit c can be reconstituted into liposomes using a 4:1 mixture of phosphatidylcholine and phosphatidic acid, which has been successfully used for other bacterial ATP synthase components .

How can site-directed mutagenesis be used to study the proton-binding site in Acidovorax sp. ATP synthase subunit c?

Site-directed mutagenesis of Acidovorax sp. ATP synthase subunit c provides a powerful approach to elucidate the functional importance of specific residues in proton binding and translocation. A comprehensive mutagenesis strategy should focus on:

Key Residues for Mutation:

• The conserved glutamate/aspartate in the C-terminal helix (equivalent to Glu61 in M. tuberculosis)

• Residues surrounding the ion-binding site that may contribute to proton affinity

• Residues at the interface between adjacent c subunits that form the complete binding pocket

Recommended Methodological Approach:

• Create mutations using overlap extension PCR or QuikChange site-directed mutagenesis

• Clone mutated genes into an expression vector with the wild-type atpE gene as control

• Express and purify both wild-type and mutant proteins

• Reconstitute proteins into liposomes for functional assays

• Assess proton translocation efficiency using pH-sensitive fluorescent dyes

• Measure ATP synthesis activity in a reconstituted system

Mutations that have proven informative in other bacterial species include Glu→Gln at the proton-binding site (eliminates proton binding), Asp→Ala at positions interacting with the proton-binding site (alters binding kinetics), and mutations at the interface between subunits (affects c-ring stability) . For Acidovorax sp., mutations equivalent to the Asp28→Gly/Ala, Glu61→Asp, and Ala63→Pro found in Mycobacterium species would be particularly valuable to investigate species-specific differences in function .

What analytical techniques are most effective for characterizing the oligomeric state of recombinant Acidovorax sp. ATP synthase subunit c?

The oligomeric state of the Acidovorax sp. ATP synthase C-ring is crucial for understanding its function and can be characterized using several complementary techniques:

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE):

• Solubilize purified protein in mild detergent (0.5-1% digitonin)

• Separate complexes on 4-16% gradient native gels with Coomassie G-250

• Use known molecular weight standards for size estimation

• Western blot with anti-His antibodies to confirm identity

Analytical Ultracentrifugation:

• Perform sedimentation velocity experiments at multiple protein concentrations

• Analyze data using SEDFIT software to determine sedimentation coefficients

• Calculate molecular weight to determine oligomeric state

• Compare results with known bacterial C-rings (8-15 subunits)

Transmission Electron Microscopy (TEM) with Negative Staining:

• Apply purified C-rings to glow-discharged carbon-coated grids

• Stain with uranyl acetate or phosphotungstic acid

• Image at magnification of 40,000-60,000×

• Perform particle analysis to determine ring diameter and subunit number

Cross-linking Mass Spectrometry:

• Treat purified protein with membrane-permeable cross-linkers (DSS or BS3)

• Digest cross-linked complexes with trypsin

• Analyze peptides by LC-MS/MS

• Identify cross-linked residues to map subunit interfaces

These techniques should be used in combination, as each provides different but complementary information about the C-ring structure. For accurate assessment, researchers should compare results with known bacterial C-ring structures from species such as Ilyobacter tartaricus, Bacillus pseudofirmus, or Spirulina platensis, which have 11, 13, and 15 subunits respectively .

What are the recommended approaches for studying proton translocation through recombinant Acidovorax sp. ATP synthase subunit c?

Investigating proton translocation through the Acidovorax sp. ATP synthase c-subunit ring requires specialized techniques that maintain the protein's native environment while allowing measurement of proton movement. The following methodological approaches are recommended:

Liposome Reconstitution System:

• Purify recombinant Acidovorax sp. c-subunit or complete ATP synthase complex

• Prepare liposomes using E. coli polar lipids or synthetic lipids (POPC:POPG at 3:1 ratio)

• Incorporate purified protein using detergent-mediated reconstitution

• Remove detergent using Bio-Beads or dialysis

• Verify reconstitution by freeze-fracture electron microscopy

pH-Sensitive Fluorescence Measurements:

• Entrap pH-sensitive fluorescent dyes (ACMA or pyranine) in proteoliposomes

• Establish a pH gradient across the membrane using buffer exchange

• Monitor fluorescence changes upon addition of ionophores or ATP

• Calculate proton translocation rates under different conditions

• Compare wild-type with mutant versions to identify critical residues

Patch-Clamp Electrophysiology:
For direct measurement of proton currents, researchers can form "artificial membranes" containing the reconstituted c-subunit ring:

• Form planar lipid bilayers across a small aperture

• Incorporate purified c-subunit rings

• Apply voltage and measure currents using high-sensitivity amplifiers

• Determine conductance and ion selectivity

• Test effects of specific inhibitors

Electrochemical Detection:

• Use solid-supported membrane (SSM) electrophysiology

• Adsorb proteoliposomes onto the SSM

• Apply rapid substrate concentration jumps

• Measure capacitive currents resulting from charge translocation

These techniques should be performed under varying conditions (pH, temperature, membrane potential) to fully characterize the proton translocation properties of the Acidovorax sp. c-subunit ring and how they might differ from other bacterial species .

How can inhibitor binding studies with Acidovorax sp. ATP synthase subunit c inform structure-based drug design?

Inhibitor binding studies with Acidovorax sp. ATP synthase subunit c can provide valuable information for structure-based drug design, particularly for developing species-selective antimicrobial compounds. A systematic approach should include:

Binding Site Identification:

• Perform homology modeling of Acidovorax sp. c-subunit based on known bacterial structures

• Use computational docking to predict binding pockets

• Identify residues likely involved in inhibitor interactions

• Compare with binding sites in other species to identify unique features

Experimental Binding Assays:

• Surface plasmon resonance (SPR) with immobilized c-subunit

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Fluorescence-based binding assays using environment-sensitive probes

• Competition binding assays with known inhibitors

Structure-Activity Relationship Studies:

• Test a series of structural analogs of known ATP synthase inhibitors

• Measure IC50 values against purified Acidovorax sp. ATP synthase

• Correlate structural features with inhibitory potency

• Develop a pharmacophore model specific to Acidovorax sp.

Table 1: Comparison of Inhibitor Binding to ATP Synthase Subunit c from Different Species

*Values for Acidovorax sp. are estimated based on related bacterial species and would need experimental verification

For structure-based drug design, researchers should focus on the key differences between Acidovorax sp. and other species. Based on studies with Mycobacterium, the cleft located between two adjacent c subunits that encompasses the proton-binding site would be a primary target. In Mycobacterium, this region includes residues Glu61, Tyr64, and Asp28, which anchor TMC207 through ionic, hydrogen, and halogen bonds respectively . Identifying the equivalent residues in Acidovorax sp. and how they differ structurally would enable the design of species-selective inhibitors.

What techniques can be used to study the interaction between Acidovorax sp. ATP synthase subunit c and other components of the ATP synthase complex?

Understanding the interactions between Acidovorax sp. ATP synthase subunit c and other components of the ATP synthase complex requires a combination of biochemical, biophysical, and structural approaches. The following methodological framework is recommended:

Co-purification and Pull-down Assays:

• Express recombinant subunit c with an affinity tag

• Express other ATP synthase components (a, b, δ, ε) with different tags

• Perform pull-down experiments to identify stable interactions

• Analyze complexes by SDS-PAGE and Western blotting

• Quantify binding affinities using varying salt concentrations

Cross-linking Mass Spectrometry:

• Treat purified ATP synthase or membrane fractions with chemical cross-linkers

• Digest cross-linked proteins with trypsin

• Enrich cross-linked peptides using SEC or SCX chromatography

• Analyze by high-resolution LC-MS/MS

• Identify cross-linked residues to map interaction interfaces

FRET-based Interaction Studies:

• Generate fusion proteins with fluorescent proteins (e.g., CFP-subunit c and YFP-subunit a)

• Express in a suitable system (E. coli or Acidovorax if possible)

• Measure FRET efficiency to determine proximity and orientation

• Use acceptor photobleaching to confirm specific interactions

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

• Expose purified complexes to D₂O buffer for varying time periods

• Quench the exchange reaction and digest proteins

• Analyze deuterium incorporation by LC-MS

• Identify regions with reduced exchange rates, indicating protein-protein interfaces

Table 2: Predicted Interaction Interfaces Between Subunit c and Other ATP Synthase Components

*Residue numbers are approximate and would need to be determined specifically for Acidovorax sp.

These techniques, used in combination, can provide a comprehensive understanding of how the c-subunit interacts with other components to form a functional ATP synthase complex in Acidovorax sp. Researchers should focus particularly on the c-a interface, which is critical for proton translocation, and the c-c interface, which determines the stability and functional properties of the c-ring .

How can researchers overcome challenges in expressing and purifying functional Acidovorax sp. ATP synthase subunit c?

Expressing and purifying membrane proteins like ATP synthase subunit c presents several challenges due to their hydrophobic nature. Here are systematic approaches to overcome common issues when working with Acidovorax sp. ATP synthase subunit c:

Challenge 1: Poor Expression Levels
Solution methods:

• Optimize codon usage for the expression host (E. coli)

• Test multiple promoter strengths (T7, trc, ara)

• Use specialized strains designed for membrane proteins (C41/C43(DE3), Lemo21(DE3))

• Lower expression temperature to 16-18°C

• Reduce inducer concentration (0.1-0.2 mM IPTG)

• Add stabilizing agents to culture medium (5-10% glycerol, 1% glucose)

Challenge 2: Protein Aggregation/Inclusion Bodies
Solution methods:

• Fuse with solubility-enhancing tags (MBP, SUMO)

• Co-express with chaperones (GroEL/GroES, DnaK/DnaJ)

• Add mild detergents to lysis buffer (0.1% DDM or LDAO)

• Use gentle cell disruption methods (enzymatic lysis or osmotic shock)

• If inclusion bodies form, develop refolding protocols with gradual detergent introduction

Challenge 3: Low Purification Yield
Solution methods:

• Optimize detergent type and concentration for extraction

  • Test DDM (0.5-1%), LDAO (0.5-1%), digitonin (0.5-1%)

• Add lipids during purification (0.1-0.5 mg/ml E. coli lipids)

• Include stabilizing additives in all buffers:

  • Glycerol (5-10%)

  • Cholesterol hemisuccinate (0.01-0.05%)

  • Specific phospholipids (0.1-0.5 mg/ml)

• Use tandem affinity tags for higher purity

• Perform purification at 4°C to minimize degradation

Challenge 4: Loss of Function During Purification
Solution methods:

• Preserve native lipid environment:

  • Extract with styrene-maleic acid copolymer (SMA) to form native nanodiscs

  • Use amphipols for detergent-free handling after extraction

• Add ATP synthase inhibitor during purification to stabilize conformation

• Validate function at each purification step with proton translocation assays

• Consider on-column refolding during purification

• Immediately reconstitute into liposomes after purification

Table 3: Detergent Screening for Optimal Solubilization of Acidovorax sp. ATP Synthase Subunit c

*Rating scale: + (poor) to ++++ (excellent)

By systematically addressing these challenges, researchers can significantly improve the yield and quality of purified Acidovorax sp. ATP synthase subunit c for functional and structural studies .

What controls and validation experiments are essential when studying the effects of mutations in Acidovorax sp. ATP synthase subunit c?

Essential Controls for Mutagenesis Studies:

• Wild-type Controls:

  • Express and purify wild-type protein in parallel with mutants

  • Process all samples identically to eliminate method-based artifacts

  • Include wild-type in every functional assay as reference point

• Expression Level Verification:

  • Quantify protein expression by Western blot analysis

  • Use C-terminal tags that don't interfere with function

  • Normalize functional data to expression levels

• Protein Folding and Stability Controls:

  • Perform circular dichroism (CD) spectroscopy to compare secondary structure

  • Conduct thermal shift assays to assess protein stability

  • Use limited proteolysis to verify similar folding patterns

• Multiple Mutation Types:

  • Create conservative mutations (e.g., Glu→Asp) and more severe mutations (e.g., Glu→Ala)

  • Analyze charge-preserving vs. charge-altering mutations

  • Generate complementary mutations that restore function in double mutants

Validation Experimental Approaches:

• Structure Validation:

  • Perform size-exclusion chromatography to verify oligomeric state

  • Use negative-stain electron microscopy to confirm C-ring formation

  • Validate membrane insertion using protease protection assays

• Functional Validation:

  • Measure proton translocation using multiple methodologies:

    • pH-sensitive fluorescent dyes

    • Membrane potential-sensitive dyes

    • Direct electrophysiological recordings

  • Assess ATP synthesis/hydrolysis coupling with reconstituted systems

  • Measure growth complementation in ATP synthase-deficient strains

• Binding Site Validation:

  • Conduct inhibitor binding studies with known ATP synthase inhibitors

  • Perform cross-linking experiments to validate interaction interfaces

  • Use molecular dynamics simulations to assess conformational effects

Table 4: Recommended Validation Experiments for Different Types of Mutations

These controls and validation experiments establish a robust framework for interpreting the effects of mutations in Acidovorax sp. ATP synthase subunit c, ensuring that observed phenotypes are directly attributable to the introduced mutations rather than experimental artifacts .

How might Acidovorax sp. ATP synthase subunit c contribute to understanding membrane-embedded ion channels and leaks?

Recent research indicates that ATP synthase subunit c may have broader implications for understanding membrane-embedded ion channels and leak mechanisms. For Acidovorax sp. ATP synthase subunit c, this presents several promising research directions:

Investigation of Potential Leak Mechanisms:
The ATP synthase c-subunit ring has been implicated in forming or contributing to the mitochondrial permeability transition pore (mPTP) . Similar leak mechanisms may exist in bacterial systems including Acidovorax sp. Researchers should:

• Examine c-subunit ring stability under different conditions (pH, membrane potential)

• Investigate whether isolated Acidovorax sp. c-rings can form ion-conducting pores in artificial membranes

• Measure ion selectivity and conductance properties of these potential pores

• Compare leak properties with those observed in other bacterial and mitochondrial systems

• Identify residues that influence leak probability through systematic mutagenesis

Structure-Function Relationships:
Understanding how the c-ring structure relates to ion channel-like properties could provide insights into fundamental principles of ion channel design:

• Determine whether the central pore of the c-ring can conduct ions under certain conditions

• Map the electrostatic potential throughout the c-ring structure

• Identify residues that contribute to ion selectivity

• Examine how lipid composition affects channel-like properties

• Investigate whether post-translational modifications alter leak tendency

Comparative Analyses:
Acidovorax sp. ATP synthase subunit c could serve as a model system for comparative studies:

• Compare properties with c-subunits from organisms that thrive in extreme environments

• Analyze differences between species that show varied energy coupling efficiencies

• Examine evolutionary conservation of residues associated with leak functions

• Study how environmental adaptations affect c-ring stability and leak propensity

These investigations could reveal how fundamental ion channel properties evolved in membrane-embedded protein complexes and provide insights into the dual role of ATP synthase components in both energy production and membrane permeability regulation . Understanding these mechanisms in Acidovorax sp. might reveal species-specific adaptations that could inform biomimetic applications in synthetic biology.

What computational approaches can advance our understanding of Acidovorax sp. ATP synthase subunit c dynamics and function?

Advanced computational approaches offer powerful tools for investigating the dynamics and function of Acidovorax sp. ATP synthase subunit c at atomic and molecular levels. The following methodological framework is recommended:

Homology Modeling and Structural Prediction:

• Generate multiple homology models using templates from different bacterial species

• Refine models through energy minimization and molecular dynamics

• Validate models using ProSA, PROCHECK, and Verify3D

• Submit models to AlphaFold Multimer for potential refinement

• Predict the complete C-ring structure with accurate subunit interfaces

Molecular Dynamics Simulations:

• Embed modeled C-ring in explicit lipid bilayers resembling Acidovorax sp. membrane composition

• Perform long-timescale (>1 μs) simulations to capture conformational changes

• Apply enhanced sampling techniques (metadynamics, umbrella sampling) to observe rare events

• Simulate proton transfer events using QM/MM approaches

• Analyze water wire formation and proton pathways through the complex

Ion/Proton Permeation Calculations:

• Calculate potential of mean force (PMF) profiles for proton movement

• Apply computational electrophysiology to determine conductance properties

• Estimate pKa values of key residues under different conditions

• Model the effect of membrane potential on proton translocation

• Simulate interactions with known inhibitors to identify binding modes

Coarse-Grained Simulations:

• Develop Martini-based coarse-grained models of the entire ATP synthase complex

• Simulate c-ring rotation coupled to proton translocation

• Investigate protein-lipid interactions with specific membrane compositions

• Model ATP synthase assembly dynamics and subunit interactions

• Simulate long-timescale events (>100 μs) inaccessible to all-atom approaches

Table 5: Computational Methods for Studying ATP Synthase Subunit c Properties

These computational approaches, used in combination with experimental techniques, can provide unprecedented insights into the dynamic behavior of Acidovorax sp. ATP synthase subunit c, revealing functional mechanisms that may be difficult to capture experimentally .

What are the most significant unresolved questions about Acidovorax sp. ATP synthase subunit c that merit future research?

Despite advances in our understanding of ATP synthase, several significant questions about Acidovorax sp. ATP synthase subunit c remain unresolved and merit focused research efforts. These questions span structural, functional, and evolutionary domains:

Structural Questions:

• What is the exact stoichiometry of the Acidovorax sp. c-ring, and how does it compare to other bacterial species?

• Are there species-specific structural adaptations that optimize ATP synthase function for Acidovorax's ecological niche?

• How does the structure of the c-ring respond to changes in membrane potential and pH?

• What is the precise architecture of the interface between the c-ring and other ATP synthase components in Acidovorax sp.?

Functional Questions:

• Does the Acidovorax sp. c-subunit ring exhibit ion-leak properties similar to those observed in mitochondrial systems ?

• How do the proton translocation kinetics of Acidovorax sp. ATP synthase compare to other bacterial species, and what structural features account for any differences?

• Are there regulatory mechanisms that modulate c-ring function in response to environmental conditions?

• Does the Acidovorax sp. c-ring display unique inhibitor binding properties that could be exploited for antimicrobial development?

Evolutionary Questions:

• How have the functional properties of ATP synthase subunit c evolved in Acidovorax sp. compared to related species?

• Are there horizontal gene transfer events in the evolutionary history of Acidovorax sp. atpE that have influenced its function?

• How has co-evolution with other ATP synthase subunits shaped the structure and function of subunit c?

• What selective pressures have driven the conservation or divergence of key residues in Acidovorax sp. ATP synthase subunit c?