Recombinant Delftia acidovorans ATP synthase subunit c (atpE)

What is Delftia acidovorans ATP synthase subunit c (atpE)?

Delftia acidovorans ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex, specifically within the F0 sector that spans the membrane. This 82-amino acid protein (UniProt ID: A9BPU2) functions as a key element in the proton-conducting portion of ATP synthase. The protein is encoded by the atpE gene (locus Daci_0415) and has several alternative names including ATP synthase F(0) sector subunit c, F-type ATPase subunit c, and lipid-binding protein . The full amino acid sequence is: MENILGLVALACGLIVGLGAIGASIGIALMGGKFLESSARQPELINELQTKMFILAGLID AAFLIGVAIALLFAFANPFVLA, revealing its predominantly hydrophobic nature consistent with its membrane-embedded function .

How is recombinant Delftia acidovorans atpE typically expressed?

Recombinant D. acidovorans atpE is typically expressed in Escherichia coli expression systems. The full-length protein (amino acids 1-82) is commonly produced with an N-terminal histidine tag to facilitate purification . The expression constructs usually incorporate the complete coding sequence into suitable expression vectors with strong promoters (often T7-based systems). After transformation into competent E. coli cells, expression is induced, followed by cell lysis and purification via affinity chromatography utilizing the histidine tag . The purified protein is often supplied as a lyophilized powder with purity greater than 90% as determined by SDS-PAGE analysis .

What storage conditions are recommended for recombinant D. acidovorans atpE?

For optimal stability, recombinant D. acidovorans atpE should be stored at -20°C or -80°C upon receipt, with aliquoting recommended for multiple use scenarios to avoid repeated freeze-thaw cycles . The protein is typically provided in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 or a Tris-based buffer with 50% glycerol . For working solutions, the protein can be safely stored at 4°C for up to one week . Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with addition of 5-50% glycerol (final concentration) recommended for long-term storage .

How does D. acidovorans atpE compare structurally with ATP synthase subunit c from other bacterial species?

D. acidovorans atpE exhibits the characteristic structural features of bacterial ATP synthase subunit c proteins, with highly conserved regions that are critical for proton translocation and ATP synthesis. When compared to other bacterial homologs, it maintains the canonical hairpin-like structure with two membrane-spanning α-helical domains connected by a polar loop. The protein contains the essential acidic residue (typically Asp or Glu) in the C-terminal helix that is critical for proton binding during the catalytic cycle.

Key comparative features include:

The sequence conservation typically centers around the functional domains responsible for proton translocation, while variability is more common in regions that interface with other subunits of the ATP synthase complex or adapt to specific membrane environments .

What roles might D. acidovorans atpE play in the organism's environmental adaptations?

D. acidovorans is notable for its environmental versatility, particularly its ability to survive in contaminated soils and degrade xenobiotic compounds. The ATP synthase complex, including the atpE subunit, likely plays several critical roles in these adaptations:

• Energy coupling in challenging environments: The ATP synthase must maintain functionality across varying pH and temperature conditions encountered in contaminated soils.

• Support for biodegradation activities: Studies have identified D. acidovorans strains with potential biodegradation activity toward perfluoroalkyl substances (PFAS) and other organofluorine compounds . The energy provided by ATP synthase is essential for powering these metabolic processes.

• Biofilm formation support: D. acidovorans forms biofilms that demonstrate tolerance to antimicrobials like chlorhexidine . The ATP synthase provides energy for biofilm development and maintenance.

• Adaptation to oxidative stress: In contaminated environments, D. acidovorans faces oxidative stress, requiring energy-intensive detoxification systems supported by ATP synthase activity.

These environmental adaptations may explain some of the unique sequence features of D. acidovorans atpE compared to homologs from other bacteria, potentially reflecting specific evolutionary pressures experienced by this organism .

How can recombinant D. acidovorans atpE contribute to studies on ATP synthase inhibitors and antimicrobial development?

Recombinant D. acidovorans atpE offers a valuable model system for studying ATP synthase inhibitors and potential antimicrobial compounds due to several factors:

• D. acidovorans has emerged as a clinically relevant organism, with infections reported particularly in immunocompromised patients . Understanding its ATP synthase structure and function could inform targeted antimicrobial strategies.

• The recombinant protein can be used in biochemical assays to screen potential inhibitors that specifically target the c-ring of ATP synthase, an approach that has proven successful for developing antimycobacterial agents targeting the same subunit in Mycobacterium tuberculosis.

• Structure-function studies using site-directed mutagenesis of the recombinant protein can identify critical residues for inhibitor binding, helping to design more specific antimicrobial compounds.

• The protein can be incorporated into liposomes or nanodiscs to create functional ATP synthase models for assessing how potential inhibitors affect proton translocation and ATP synthesis in a membrane environment.

• Comparative studies between D. acidovorans atpE and homologs from human mitochondria can help identify structural differences that could be exploited to develop selective antimicrobials with minimal host toxicity .

What are the optimal conditions for reconstituting and solubilizing recombinant D. acidovorans atpE?

Reconstitution and solubilization of recombinant D. acidovorans atpE requires careful attention to its hydrophobic nature as a membrane protein. The optimal protocol includes:

• Initial preparation: Briefly centrifuge the lyophilized protein vial to bring contents to the bottom before opening .

• Primary reconstitution: Dissolve the lyophilized powder in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For membrane protein studies, this initial reconstitution may require the addition of mild detergents.

• Buffer optimization: For functional studies, reconstitution in a Tris-based buffer (pH 7.5-8.0) containing physiologically relevant ions (K+, Na+, Mg2+) is recommended.

• Stabilization additives: Addition of 5-50% glycerol as a final concentration helps stabilize the protein structure. The standard recommended final glycerol concentration is 50% .

• Membrane incorporation: For functional studies, the protein can be incorporated into liposomes composed of E. coli polar lipid extract or synthetic lipid mixtures (typically 3:1 POPE:POPG) using detergent-mediated reconstitution followed by detergent removal via dialysis or Bio-Beads.

• Verification of proper folding: Circular dichroism spectroscopy can confirm the expected high alpha-helical content of properly reconstituted atpE.

• Storage of reconstituted samples: Working aliquots can be stored at 4°C for up to one week, while longer-term storage requires -20°C or -80°C .

What assays can be used to evaluate the functional activity of recombinant D. acidovorans atpE?

Several complementary approaches can be used to assess the functional activity of recombinant D. acidovorans atpE:

• Proton translocation assays:

  • pH-sensitive fluorescent dyes (ACMA or pyranine) can monitor proton movement across liposomes containing reconstituted atpE

  • Patch-clamp electrophysiology can directly measure proton currents through reconstituted c-rings in planar lipid bilayers

• ATP synthesis/hydrolysis coupling:

  • When reconstituted with other ATP synthase subunits, ATP synthesis can be measured using luciferase-based ATP detection assays

  • Proton-pumping activity can be measured by creating an artificial proton gradient and monitoring ATP hydrolysis rates

• Binding assays:

  • Isothermal titration calorimetry (ITC) can quantify binding of inhibitors or other ligands

  • Surface plasmon resonance (SPR) can determine binding kinetics of interactions with other subunits or inhibitors

• Structural integrity assessment:

  • Circular dichroism spectroscopy confirms proper secondary structure (predominantly alpha-helical)

  • Limited proteolysis followed by mass spectrometry verifies the expected membrane topology

• Oligomerization analysis:

  • Native gel electrophoresis can assess c-ring formation

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) can determine the oligomeric state of the c-ring complex

Each of these methods provides complementary information about different aspects of atpE function, from individual protein conformation to integration into the complete ATP synthase complex .

What are the key considerations when designing mutagenesis studies of D. acidovorans atpE?

When designing mutagenesis studies for D. acidovorans atpE, researchers should consider the following critical factors:

Carefully designed mutagenesis studies can provide valuable insights into structure-function relationships of D. acidovorans atpE and its role in ATP synthesis and bacterial physiology .

How can researchers differentiate between specific effects on atpE function versus general membrane disruption in inhibitor studies?

Distinguishing between specific inhibition of atpE function and non-specific membrane disruption is crucial when evaluating potential ATP synthase inhibitors. A comprehensive approach includes:

• Parallel membrane integrity assessments:

  • Measure liposome permeability using calcein release assays alongside functional studies

  • Monitor membrane fluidity changes using fluorescence anisotropy with probes like DPH

  • Assess membrane potential using voltage-sensitive dyes (e.g., DiSC3(5)) to detect non-specific effects

• Concentration-response relationships:

  • Specific inhibitors typically show defined concentration-response curves with clear saturation

  • Membrane-disrupting agents often display steep curves without clear saturation

  • Compare IC50 values for ATP synthase inhibition versus membrane disruption metrics

• Competitive binding studies:

  • Test if known c-subunit ligands can competitively reverse inhibition

  • Utilize direct binding assays (ITC, SPR) to confirm specific interaction with purified protein

• Mutational validation:

  • Generate point mutations in predicted binding sites to confirm specificity

  • A true inhibitor should show altered potency against specific mutants

  • Non-specific membrane disruptors typically retain activity regardless of protein mutations

• Cross-validation with other membrane proteins:

  • Test effects on unrelated membrane proteins or transporters

  • Specific inhibitors should not affect function of unrelated membrane proteins at concentrations that inhibit atpE

• Time-course analysis:

  • Specific inhibition often shows defined kinetics

  • Membrane disruption typically produces rapid, sometimes irreversible effects

By implementing this multi-faceted approach, researchers can confidently differentiate between compounds that specifically target D. acidovorans atpE and those that simply disrupt membrane integrity, leading to more reliable characterization of potential ATP synthase inhibitors .

What challenges arise when comparing D. acidovorans atpE with homologs from other bacterial species, and how can these be addressed?

Comparative analysis of ATP synthase subunit c across bacterial species presents several challenges that require careful methodological approaches:

Addressing these challenges requires an integrated approach combining sequence analysis, structural studies, and functional assays normalized for the specific properties of each homolog. This enables more reliable interpretation of observed differences and their evolutionary or functional significance .

What statistical approaches are most appropriate for analyzing comparative studies of wild-type versus mutant D. acidovorans atpE?

• For enzymatic activity and kinetic parameters:

  • Michaelis-Menten kinetics analysis for determining Km and Vmax changes

  • Statistical significance of differences should be assessed using Student's t-test (for comparing two variants) or ANOVA with post-hoc tests (for multiple variants)

  • Effect size calculations (Cohen's d) help quantify the magnitude of functional changes

  • Non-linear regression for fitting more complex kinetic models, with comparison of model fit using AIC or BIC criteria

• For binding studies:

  • Analyze binding isotherms using appropriate models (one-site, two-site, cooperative binding)

  • Compare dissociation constants (Kd) and thermodynamic parameters (ΔH, ΔS, ΔG) using paired statistical tests

  • For SPR data, compare kon and koff rates using bootstrap resampling techniques

• For structural stability measurements:

  • Thermal denaturation curves should be analyzed using sigmoidal fitting to extract Tm values

  • Compare structural parameters from CD spectroscopy using multivariate analysis to account for interdependencies

• For proton translocation assays:

  • Time-course data should be analyzed using area-under-curve (AUC) approaches or by fitting to exponential models

  • Rate constants can be compared using paired statistical tests

• Appropriate experimental design considerations:

  • Ensure adequate technical replicates (minimum n=3) and biological replicates (different protein preparations)

  • Use blocked experimental designs to control for batch effects

  • Include appropriate positive and negative controls in each experimental batch

• Dealing with outliers and variability:

  • Apply Grubb's test or other outlier detection methods before analysis

  • Consider non-parametric tests (Mann-Whitney U, Kruskal-Wallis) when data violate normality assumptions

  • Use robust statistical methods resistant to outliers and heteroscedasticity

• Multiple testing correction:

  • When testing multiple mutants or conditions, apply Bonferroni or false discovery rate corrections

  • Consider global testing approaches before post-hoc comparisons

How might D. acidovorans atpE research contribute to understanding the organism's role in biodegradation of environmental contaminants?

D. acidovorans has shown promising capabilities for biodegradation of environmental pollutants, including perfluoroalkyl substances (PFAS) and other organofluorine compounds . Research on its ATP synthase subunit c (atpE) could provide critical insights into these processes:

• Energy coupling in degradation pathways:

  • ATP synthase supplies the energy required for resource-intensive biodegradation processes

  • Investigation of atpE function under conditions mimicking contaminated environments could reveal adaptations that support biodegradation

  • Research could examine how the ATP synthase complex maintains functionality in the presence of contaminants that might disrupt membrane integrity

• Biofilm formation and maintenance:

  • D. acidovorans forms biofilms that enhance its biodegradation capabilities and antimicrobial tolerance

  • Studies could explore how ATP synthesis supports the energetic requirements of biofilm formation

  • Research might investigate if atpE variants correlate with enhanced biofilm formation in strains with superior biodegradation capabilities

• Adaptation to extreme environments:

  • Comparative studies of atpE from D. acidovorans strains isolated from differently contaminated environments could reveal adaptive variations

  • Research could examine how the ATP synthase complex maintains functionality across varying pH conditions encountered in contaminated soils

  • Investigations might explore potential interactions between atpE and dehalogenase enzymes identified in biodegradation-capable strains

• Metabolic integration with dehalogenation pathways:

  • Studies could explore the energetic coupling between ATP synthesis and the recently characterized dehalogenases in D. acidovorans (DeHa1-5)

  • Research might investigate how ATP synthase activity supports or limits the rate of dehalogenation reactions

  • Experiments could test if inhibition of ATP synthase affects the organism's ability to degrade environmental contaminants

• Potential biotechnological applications:

  • Knowledge of atpE function could inform genetic engineering approaches to enhance D. acidovorans biodegradation capabilities

  • Research might explore creating optimized strains with modified ATP synthase properties for bioremediation applications

  • Studies could investigate coupling ATP synthesis to specific biodegradation pathways for improved efficiency

These research directions could significantly advance our understanding of how D. acidovorans' energy metabolism supports its environmental adaptations and biodegradation capabilities .

What emerging technologies might advance structural and functional studies of D. acidovorans atpE?

Several cutting-edge technologies hold promise for deepening our understanding of D. acidovorans atpE structure and function:

• Cryo-electron microscopy (cryo-EM) advances:

  • Single-particle cryo-EM now achieves near-atomic resolution for membrane proteins

  • Application to D. acidovorans ATP synthase could reveal the complete c-ring structure

  • Time-resolved cryo-EM might capture conformational changes during the catalytic cycle

• Integrative structural biology approaches:

  • Combining X-ray crystallography, NMR, cryo-EM, and computational modeling

  • Cross-linking mass spectrometry (XL-MS) to map interaction interfaces within the ATP synthase complex

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify dynamic regions

• Advanced single-molecule techniques:

  • Single-molecule FRET to track conformational changes during proton translocation

  • Magnetic tweezers or optical traps to measure mechanical forces during ATP synthesis

  • Nanopore recordings of individual c-rings in lipid bilayers

• In-cell structural biology:

  • Cellular cryo-electron tomography to visualize ATP synthase in its native environment

  • In-cell NMR to detect conformational states in living cells

  • Proximity labeling techniques (BioID, APEX) to map the protein interaction network in vivo

• Artificial intelligence and computational approaches:

  • AlphaFold2 and RoseTTAFold for accurate structure prediction

  • Molecular dynamics simulations with enhanced sampling to model proton transfer

  • Machine learning analysis of sequence-function relationships across bacterial homologs

• Novel membrane mimetics:

  • Nanodiscs with defined lipid compositions to study lipid-protein interactions

  • Cell-derived giant plasma membrane vesicles (GPMVs) to maintain native membrane context

  • Microfluidic systems for rapid testing of membrane protein function in different environments

• Gene editing and high-throughput functional assays:

  • CRISPR-Cas9 editing of D. acidovorans to create comprehensive mutation libraries

  • Deep mutational scanning to comprehensively map sequence-function relationships

  • Microfluidic single-cell assays to correlate ATP synthase activity with cellular phenotypes

These technological advances could revolutionize our understanding of ATP synthase structure and function, particularly for environmentally important organisms like D. acidovorans .

How might understanding D. acidovorans atpE contribute to addressing antibiotic resistance concerns associated with this organism?

Recent clinical reports have identified D. acidovorans as an emerging opportunistic pathogen, particularly in healthcare settings . Understanding its ATP synthase subunit c (atpE) could contribute significantly to addressing antibiotic resistance concerns:

• Development of targeted antimicrobials:

  • ATP synthase is essential for bacterial survival and represents an attractive drug target

  • Structural knowledge of D. acidovorans atpE could enable design of specific inhibitors

  • Comparative analysis with human mitochondrial ATP synthase could identify bacterial-specific targets to minimize toxicity

  • Previous success targeting mycobacterial ATP synthase (e.g., bedaquiline) provides proof-of-concept

• Understanding resistance mechanisms:

  • Studies could identify potential mutations in atpE that might confer resistance to ATP synthase inhibitors

  • Research might explore if clinical isolates show variations in atpE sequence or expression

  • Investigation of potential compensatory mechanisms when ATP synthase is inhibited could inform combination therapy approaches

• Biofilm-associated resistance:

  • D. acidovorans forms biofilms that demonstrate tolerance to antimicrobials including chlorhexidine

  • Research could examine how ATP synthase function supports biofilm formation and maintenance

  • Studies might investigate if targeting ATP synthase disrupts biofilm integrity, potentially re-sensitizing bacteria to conventional antibiotics

• Metabolic vulnerabilities:

  • ATP synthase inhibition creates metabolic perturbations that might be exploited

  • Research could explore synergistic drug combinations targeting both ATP synthesis and compensatory pathways

  • Studies might investigate metabolic bottlenecks specific to D. acidovorans that could be therapeutic targets

• Host-pathogen interaction insights:

  • ATP synthesis requirements during infection might differ from laboratory conditions

  • Research could examine atpE expression and function during host cell interaction

  • Studies might investigate if host immune factors specifically target ATP synthase components

• Diagnostic applications:

  • Specific molecular signatures in atpE might enable rapid identification of D. acidovorans in clinical samples

  • Understanding functional variations in atpE across strains could help identify particularly virulent variants

  • Research might develop ATP synthase activity assays as indicators of antibiotic susceptibility

These research directions could significantly advance our ability to combat D. acidovorans infections, particularly in vulnerable patient populations where this organism has been identified as an emerging concern .