Recombinant Thiobacillus denitrificans ATP synthase subunit c (atpE)

What is the structural role of subunit c in T. denitrificans ATP synthase?

Subunit c (atpE) in T. denitrificans ATP synthase forms part of the membrane-embedded F₀ domain, which is responsible for proton translocation across the membrane. This subunit assembles into a ring structure that rotates during ATP synthesis and hydrolysis. While the search results don't specifically detail the T. denitrificans c-subunit structure, the general architecture of ATP synthase in this organism includes several regulatory components that interact with the F₀ domain, including the ε subunit with its C-terminal domain (ε-CTD) and the unique ζ subunit .

Methodologically, structural characterization of subunit c typically employs techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy. When working with recombinant subunit c, researchers should consider detergent selection for membrane protein solubilization, as this can significantly impact structural integrity.

How does T. denitrificans ATP synthase regulation differ from other bacterial species?

T. denitrificans ATP synthase exhibits unique regulatory properties compared to other bacterial species. The enzyme demonstrates remarkably low ATP hydrolysis rates compared to synthesis rates, making it functionally unidirectional. This regulation involves multiple mechanisms:

• The ζ subunit, unique to P. denitrificans ATP synthase, was initially thought to be the primary hydrolysis inhibitor (similar to mammalian IF₁ inhibitor) .

• The ε subunit C-terminal domain (ε-CTD) plays a regulatory role but appears less significant in T. denitrificans than in other bacteria .

• Mg-ADP inhibition serves as the predominant regulatory mechanism .

Experimentally, deletion of the ζ subunit increases hydrolysis only two-fold (to 0.026 μmol min⁻¹ mg⁻¹), while deletion of the ε-CTD shows no statistically significant increase in hydrolysis rates . These rates remain far below those observed in E. coli (0.38 μmol min⁻¹ mg⁻¹) and bovine mitochondria (1.24 μmol min⁻¹ mg⁻¹) under similar conditions .

What expression systems are optimal for recombinant production of T. denitrificans atpE?

For recombinant expression of T. denitrificans atpE, researchers should consider host compatibility, membrane integration, and functional integrity. While the search results don't specifically address atpE expression, the following methodological approaches can be applied based on general principles and related work with T. denitrificans proteins:

• E. coli-based expression systems: Modified strains like C41(DE3) or C43(DE3) designed for membrane protein expression can be used with vectors containing T7 or tac promoters.

• Cell-free expression systems: These may allow better control of detergent environment for proper folding.

• Homologous expression: Expression in related species may provide proper folding machinery.

Based on work with other T. denitrificans proteins, inclusion of a purification tag (His₆) at either the N- or C-terminus with a TEV protease cleavage site allows for efficient purification while permitting tag removal for functional studies. Expression should be optimized by testing different induction temperatures (typically 18-30°C) and inducer concentrations.

How does the Mg-ADP inhibition mechanism in T. denitrificans ATP synthase affect recombinant subunit c function?

Mg-ADP inhibition represents a primary regulatory mechanism in T. denitrificans ATP synthase, with significant implications for functional studies of recombinant subunit c. Research indicates that ATP hydrolysis in T. denitrificans can be activated through three distinct methods that relieve Mg-ADP inhibition:

• Generation of proton motive force (Δp)

• Addition of the detergent lauryldimethylamine oxide (LDAO)

• Addition of oxyanions such as selenite or phosphate

ATP hydrolysis rates in μmol min⁻¹ mg⁻¹

For methodological approaches to studying recombinant subunit c in this context, researchers should design experiments that account for these activation mechanisms. Reconstitution studies should incorporate methods to measure proton translocation coupled to ATP hydrolysis both in the presence and absence of these activators. This allows distinction between effects on subunit c assembly versus regulatory effects on the intact enzyme complex.

What mutations in recombinant T. denitrificans atpE affect proton translocation and enzyme coupling?

When investigating structure-function relationships in recombinant T. denitrificans atpE, researchers should focus on residues involved in proton binding and translocation, as well as those affecting the interaction with other subunits. While the search results don't specifically address atpE mutations, the following methodological approach can be used:

• Site-directed mutagenesis targeting:

  • The conserved carboxyl residue (typically Asp or Glu) in the middle of the membrane-spanning helix

  • Residues involved in subunit-subunit interactions within the c-ring

  • Residues at the interface with the ε and ζ subunits

• Functional assessment:

  • ATP hydrolysis measurements using NADH-coupled ATP regenerating assays

  • Proton pumping measurements using pH-sensitive fluorescent dyes

  • Assessment of activation by LDAO, oxyanions, and proton motive force

Notably, studies involving other ATP synthase components reveal that removing both the ζ subunit and ε-CTD does not significantly increase ATP hydrolysis rates above the minimal levels observed in wild-type (≤0.074 μmol min⁻¹ mg⁻¹), suggesting that c-subunit mutations alone might not overcome the dominant Mg-ADP inhibition mechanism .

How does the unique hexameric assembly of T. denitrificans APS kinase relate to ATP synthase function?

While not directly related to ATP synthase subunit c, the hexameric assembly pattern observed in T. denitrificans APS kinase provides interesting structural insights that may inform our understanding of multisubunit enzyme complexes in this organism. The 2.95 Å resolution X-ray crystal structure of Tbd-0210 gene product reveals a hexameric assembly with D₃ symmetry, where each subunit contains an N-terminal sulfurylase-like domain (inactive) and a C-terminal APS kinase domain .

This hexameric assembly pattern is reminiscent of the hexameric fungal ATP sulfurylases from Penicillium chrysogenum and Saccharomyces cerevisiae, suggesting a potential evolutionary relationship or structural conservation pattern in T. denitrificans enzymes . For researchers studying recombinant T. denitrificans atpE, this observation raises methodological considerations for investigating potential oligomeric states or assembly patterns of the c-subunit ring.

When studying recombinant atpE, researchers should employ size exclusion chromatography, analytical ultracentrifugation, and native gel electrophoresis to characterize the oligomeric state and assembly kinetics. The domain organization pattern observed in the APS kinase might suggest that T. denitrificans has evolved unique structural solutions for multisubunit enzyme complexes.

What approaches can resolve contradictory data regarding ε-CTD function in T. denitrificans ATP synthase?

The search results reveal apparent contradictions in the literature regarding the role of the ε-CTD in ATP synthase regulation. While ε-CTD is a significant regulator in some bacteria (e.g., causing a 5-fold increase in activity when removed in Bacillus PS3), its removal in T. denitrificans shows no significant effect on ATP hydrolysis rates . Additionally, there are contradictory reports about whether ε-CTD removal affects proton pumping in E. coli .

To resolve these contradictions when studying recombinant T. denitrificans atpE, researchers should employ the following methodological approaches:

• Comprehensive kinetic analysis:

  • Measure ATP hydrolysis with varying ATP concentrations

  • Determine Km and Vmax values in different regulatory contexts

  • Analyze the cooperativity of ATP binding using Hill coefficients

• Direct protein-protein interaction studies:

  • Employ FRET-based approaches to monitor interactions between subunit c and regulatory subunits

  • Use cross-linking studies followed by mass spectrometry

  • Perform co-immunoprecipitation with antibodies against specific subunits

• Mixed reconstitution experiments:

  • Combine recombinant subunit c with native or modified F₁ components

  • Analyze how different combinations affect catalytic properties

A data table comparing experimental results across multiple bacterial species would help identify specific factors that might explain these contradictions:

What protocols optimize the purification of recombinant T. denitrificans atpE?

Purification of recombinant T. denitrificans atpE requires specialized approaches due to its hydrophobic nature as a membrane protein. While the search results don't specifically address atpE purification, the following methodological workflow can be applied:

• Cell lysis options:

  • French press (20,000 psi)

  • Sonication (10 cycles of 30s on/30s off)

  • Detergent-based extraction (e.g., n-dodecyl β-D-maltoside)

• Purification strategy:

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

  • Size exclusion chromatography to separate monomeric from oligomeric forms

  • Ion exchange chromatography as a polishing step

• Critical considerations:

  • Detergent selection affects stability and activity (LDAO has been shown to activate ATP hydrolysis in T. denitrificans ATP synthase)

  • Lipid supplementation may be necessary to maintain structural integrity

  • Temperature control during purification (4°C recommended)

For quality control, purified recombinant atpE should be assessed via SDS-PAGE, Western blotting, mass spectrometry, and circular dichroism to confirm identity, purity, and proper folding before functional studies.

How can researchers effectively measure coupling between ATP synthesis/hydrolysis and proton translocation in recombinant T. denitrificans ATP synthase systems?

Measuring the coupling efficiency between ATP synthesis/hydrolysis and proton translocation represents a critical aspect of functional characterization. Based on methodologies mentioned in the search results, researchers should:

• For ATP hydrolysis activity:

  • Employ NADH-coupled ATP regenerating assays as described for T. denitrificans sub-bacterial particles (SBPs)

  • Monitor activity with and without uncouplers like gramicidin to assess membrane integrity

  • Test activation by LDAO, selenite, and other oxyanions that relieve Mg-ADP inhibition

• For proton translocation:

  • Use pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Employ 9-amino-6-chloro-2-methoxyacridine (ACMA) quenching assays

  • Measure transmembrane pH gradients with dual-wavelength spectroscopy

• For coupling ratio determination:

  • Calculate the H⁺/ATP ratio by simultaneously measuring ATP hydrolysis and proton translocation

  • Compare results with and without known uncouplers

Special consideration should be given to the unique regulatory properties of T. denitrificans ATP synthase. Experiments must account for the minimal ATP hydrolysis activity under standard conditions (≤0.074 μmol min⁻¹ mg⁻¹) and the substantial activation by LDAO (up to 0.80 μmol min⁻¹ mg⁻¹ in Δζ strains) .

What reconstitution methods best preserve native-like function of recombinant T. denitrificans atpE?

For functional studies of recombinant T. denitrificans atpE, proper reconstitution into a membrane environment is crucial. While the search results don't specifically address reconstitution methods, the following methodological approaches should be considered:

• Liposome preparation options:

  • Extrusion through polycarbonate filters (100-400 nm pore size)

  • Sonication to form small unilamellar vesicles

  • Detergent dialysis method with Bio-Beads SM-2 for detergent removal

• Lipid composition considerations:

  • E. coli polar lipid extract provides a bacterial membrane-like environment

  • DOPC/DOPE/cardiolipin mixtures (70:20:10) mimic bacterial membranes

  • Native T. denitrificans lipid extraction (if possible) would be optimal

• Protein:lipid ratio optimization:

  • Test ratios ranging from 1:50 to 1:200 (w/w)

  • Monitor reconstitution efficiency via freeze-fracture electron microscopy

  • Assess orientation using proteolytic digestion of exposed regions

• Functional validation:

  • ATP-dependent proton pumping assays using pH-sensitive dyes

  • Patch-clamp electrophysiology for single-channel conductance

  • Comparison with native membrane vesicles (SBPs)

Researchers should note that the unique regulatory properties of T. denitrificans ATP synthase, particularly the strong Mg-ADP inhibition mechanism, may necessitate specific activation methods (LDAO, selenite, or proton motive force) to observe significant activity in reconstituted systems .

How can researchers address the challenge of low ATP hydrolysis activity in T. denitrificans ATP synthase for functional studies?

The extremely low ATP hydrolysis activity of T. denitrificans ATP synthase poses significant challenges for functional characterization. Even with genetic deletions of both ζ subunit and ε-CTD, ATP hydrolysis rates remain below 0.074 μmol min⁻¹ mg⁻¹, which is close to background levels . To address this challenge, researchers should implement:

• Activation strategies:

  • LDAO addition (optimal concentration 0.4% for wild-type, 0.2% for Δζ strains)

  • Oxyanion addition (selenite at 20 mM is effective)

  • Generation of proton motive force (Δp)

• Sensitivity enhancement:

  • Use more sensitive ATP hydrolysis assays (e.g., malachite green phosphate detection)

  • Increase protein concentration in assays

  • Extend measurement times to detect small changes

• Comparative analysis:

  • Run parallel assays with E. coli ATP synthase as a positive control

  • Use known activators and inhibitors to calibrate assay sensitivity

The following data table illustrates the activation potential of different treatments:

ATP hydrolysis rates in μmol min⁻¹ mg⁻¹

This considerable increase in activity with activation demonstrates that functional studies are feasible despite low basal activity.

What analytical methods can distinguish between monomeric and oligomeric forms of recombinant T. denitrificans atpE?

Distinguishing between different oligomeric states of recombinant T. denitrificans atpE is crucial for structural and functional studies. Based on techniques mentioned for analyzing T. denitrificans ATP synthase components, researchers should employ:

• Electrophoretic techniques:

  • Blue Native PAGE (BN-PAGE) as used for T. denitrificans ATP synthase complex analysis

  • Tricine-SDS-PAGE for higher resolution of low molecular weight proteins

  • Ferguson plots (using gradient gels) to estimate native molecular weight

• Chromatographic approaches:

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

  • Ion exchange chromatography under non-denaturing conditions

  • Hydrophobic interaction chromatography to separate different oligomeric forms

• Biophysical techniques:

  • Analytical ultracentrifugation (sedimentation velocity and equilibrium)

  • Dynamic light scattering for size distribution analysis

  • Chemical cross-linking followed by mass spectrometry

• Microscopy methods:

  • Negative stain electron microscopy

  • Atomic force microscopy for topological analysis

  • Single-particle cryo-electron microscopy for structural characterization

How might CRISPR-Cas9 genome editing be applied to study T. denitrificans atpE function in vivo?

CRISPR-Cas9 genome editing offers powerful approaches for investigating T. denitrificans atpE function directly in its native context. While traditional genetic manipulation has been used to create deletion mutants of regulatory components like the ζ subunit and ε-CTD , CRISPR-Cas9 could enable more precise and efficient genetic modifications. Researchers should consider:

• Methodological workflow:

  • Design of guide RNAs targeting atpE and flanking regions

  • Development of a CRISPR-Cas9 delivery system compatible with T. denitrificans

  • Use of homology-directed repair with donor templates containing desired mutations

  • Screening and verification of edited strains by sequencing

• Potential genetic modifications:

  • Introduction of point mutations in key residues involved in proton translocation

  • Creation of fluorescent protein fusions for localization studies

  • Installation of affinity tags for in vivo interaction studies

  • Generation of conditional knockdown systems

• Phenotypic characterization:

  • Growth rate analysis under various metabolic conditions

  • ATP synthesis and hydrolysis measurements in membrane vesicles

  • Proton translocation efficiency assays

  • Response to inhibitors and activators of ATP synthase

This approach would complement existing genetic methods used for T. denitrificans, such as the unmarked genetic deletions created via double homologous recombination for studying ζ subunit and ε-CTD functions .

What are the implications of T. denitrificans ATP synthase regulation for bioenergetic adaptation in extreme environments?

T. denitrificans is a chemolithoautotrophic bacterium capable of growth under denitrifying conditions, suggesting its ATP synthase has evolved to function optimally in specific environmental niches. The unique regulatory features of T. denitrificans ATP synthase, particularly its strong unidirectionality favoring synthesis over hydrolysis , have important implications for bioenergetic adaptation.

Research questions that emerge from this understanding include:

• Evolutionary considerations:

  • Has the unidirectional nature of T. denitrificans ATP synthase evolved as an energy conservation strategy?

  • How does the regulation compare with ATP synthases from other extremophiles?

  • Is the predominant Mg-ADP inhibition mechanism an adaptation to specific environmental conditions?

• Methodological approaches:

  • Comparative biochemical analysis of ATP synthases from related organisms

  • Measurement of ATP synthesis/hydrolysis ratios under varying conditions

  • Assessment of gene expression patterns under different growth conditions

• Applied research potential:

  • Engineering of unidirectional ATP synthases for biotechnological applications

  • Development of energy conservation strategies inspired by T. denitrificans

  • Design of inhibitors targeting the unique regulatory mechanisms

Future research should explore how the specific structure and function of atpE contributes to these unique regulatory properties and whether targeted modifications could alter the unidirectionality for biotechnological applications while maintaining energy efficiency.