Recombinant Leptothrix cholodnii ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Leptothrix cholodnii and how does it function?

ATP synthase subunit c (atpE) serves as a critical component of F-type ATPases in Leptothrix cholodnii, catalyzing ATP production from ADP in the presence of a proton gradient. The protein forms part of the F0 domain, functioning as the membrane-embedded proton channel that drives ATP synthesis through a rotary mechanism .

In ATP synthase complexes, subunit c molecules assemble into a cylindrical oligomeric structure (typically a c-ring) that serves as the central rotor element of the F1 domain. This structure contains residues between positions 5-25 and 57-77 that are critical for proton translocation across the membrane . The rotation of this c-ring, driven by proton flow, directly couples to the catalytic function of the F1 domain, enabling the phosphorylation of ADP to ATP.

How does Leptothrix cholodnii's unique biology influence ATP synthase function?

Leptothrix cholodnii possesses distinctive biological features that likely impact its ATP synthase function. The bacterium generates filaments encased in a sheath comprised of woven nanofibrils and forms porous pellicles at air-liquid interfaces in static liquid cultures . This specialized extracellular structure may create unique microenvironments that influence proton gradients and energy metabolism.

The nanofibril sheath has been shown to lower cell surface hydrophobicity by approximately 60%, which affects cellular aggregation and mobility at interfaces . This modified surface property could potentially influence membrane protein organization, including ATP synthase complexes. Unlike sheathless hydrophobic mutants that get trapped at interfaces, the sheathed wild-type cells retain mobility while forming interconnected networks, suggesting specialized energy requirements that would involve ATP synthase function.

What expression systems are most effective for producing recombinant Leptothrix cholodnii ATP synthase subunit c?

For optimal expression of recombinant Leptothrix cholodnii ATP synthase subunit c, a heterologous yeast expression system has proven effective for related ATP synthase components . This approach offers several advantages for membrane protein expression:

Table 1: Comparison of Expression Systems for Recombinant ATP Synthase Subunit c

For Leptothrix cholodnii ATP synthase subunit c specifically, the protein should be expressed with a cleavable affinity tag (His6 or StrepII) to facilitate purification while enabling tag removal for functional studies. Codon optimization based on the target expression system is essential, as is temperature regulation during induction (typically 18-25°C) to reduce aggregation of this highly hydrophobic protein.

What are the most reliable methods for purifying functional Leptothrix cholodnii ATP synthase subunit c?

Purification of functional Leptothrix cholodnii ATP synthase subunit c requires specialized approaches for membrane proteins:

• Membrane isolation: Following cell lysis, differential centrifugation should be performed to isolate membrane fractions (typically 100,000 × g for 1 hour).

• Detergent solubilization: Test multiple detergents for optimal solubilization while preserving protein structure:

  • Mild detergents like n-dodecyl-β-D-maltoside (DDM) at 1-2% (w/v)

  • Digitonin at 0.5-1% (w/v)

  • Styrene maleic acid lipid particles (SMALPs) for detergent-free extraction

• Affinity chromatography: Using the engineered affinity tag (His6 or StrepII) with appropriate resins.

• Size exclusion chromatography: To separate oligomeric forms and remove aggregates.

Purity assessment should be performed using SDS-PAGE (>85% purity standard) followed by immunoblotting with specific antibodies against the c subunit. For functional verification, reconstitution into proteoliposomes and measurement of proton translocation activity are essential.

How can researchers optimize storage conditions for recombinant Leptothrix cholodnii ATP synthase subunit c?

Based on storage recommendations for related ATP synthase components, the following protocol is recommended :

• Short-term storage (up to one week): Store working aliquots at 4°C in purification buffer supplemented with 0.05% DDM or appropriate detergent.

• Long-term storage: Add 5-50% glycerol (final concentration) to purified protein and store at -20°C or -80°C . Avoid repeated freeze-thaw cycles.

• Reconstitution protocol: Prior to use, briefly centrifuge the vial to bring contents to the bottom. Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL .

• Stability assessment: Monitor protein stability using circular dichroism (CD) spectroscopy before experimental use to ensure structural integrity has been maintained during storage.

What methods are most effective for measuring the enzymatic activity of recombinant Leptothrix cholodnii ATP synthase subunit c?

While isolated subunit c cannot catalyze ATP synthesis independently, its functional incorporation into complete ATP synthase complexes can be assessed through several approaches:

Direct functional assays:

• Proton pumping assay: Reconstitute purified subunit c with other ATP synthase components in proteoliposomes containing pH-sensitive fluorescent dyes (ACMA or pyranine). ATP hydrolysis-driven proton pumping can be measured as changes in fluorescence intensity.

• ATP synthesis measurement: In properly reconstituted systems, generate a proton gradient and measure ATP production using luciferase-based luminescence assays.

Structural integration assessment:

• Blue Native PAGE: To verify incorporation into ATP synthase complexes.

• Cross-linking studies: Using bifunctional cross-linkers to analyze interactions with other subunits.

• Hydrogen-deuterium exchange mass spectrometry: To assess structural dynamics and protein-protein interactions.

The efficiency of ATP synthesis in complexes containing the recombinant subunit c should be compared with native enzyme preparations to determine functional equivalence.

How does the oligomeric assembly of Leptothrix cholodnii ATP synthase subunit c differ from other bacterial homologs?

ATP synthase subunit c typically assembles into a cylindrical oligomeric c-ring structure that forms the rotor element of the complex, with the number of c subunits varying between species .

Table 2: Comparative Analysis of ATP Synthase c-ring Composition Across Species

*Predicted values based on analysis of related species and ecological niche; requires experimental verification.

The oligomeric assembly of Leptothrix cholodnii ATP synthase subunit c would likely be optimized for its unique environmental conditions, including microaerophilic habitats and interfaces between aerobic and anaerobic zones where it forms pellicles .

How can site-directed mutagenesis of Leptothrix cholodnii ATP synthase subunit c be used to study proton translocation mechanisms?

Site-directed mutagenesis of key residues in Leptothrix cholodnii ATP synthase subunit c provides valuable insights into proton translocation mechanisms. Based on conserved features of ATP synthase subunit c, the following mutations would be particularly informative:

• Essential carboxylate residue: Identify and mutate the conserved carboxylate (likely Asp or Glu) involved in proton binding. In most bacteria, this is Asp61 (E. coli numbering). Mutations to Asn, Gln, or Ala would abolish proton binding.

• Proton entry/exit channel residues: Identify residues that form the hydrophilic pathway for proton access. Mutations altering channel size or hydrophobicity would affect proton translocation kinetics.

• Inter-subunit interface residues: Mutations at the interface between adjacent c subunits can provide information about c-ring stability and rotational flexibility.

Experimental Workflow:

• Generate mutants using overlap extension PCR or CRISPR-based techniques

• Express and purify mutant proteins

• Reconstitute into liposomes with other ATP synthase components

• Measure proton translocation rates using pH-sensitive fluorophores

• Determine ATP synthesis/hydrolysis rates in reconstituted systems

• Analyze structural changes using hydrogen-deuterium exchange mass spectrometry

This approach would reveal structure-function relationships specific to Leptothrix cholodnii's ATP synthase and potentially identify unique adaptations related to its ecological niche.

What is the potential of Leptothrix cholodnii ATP synthase subunit c as a target for antimicrobial development?

ATP synthase subunit c represents a promising antimicrobial target due to its essential role in bacterial energy metabolism . For Leptothrix cholodnii specifically:

• Target validation: ATP synthase subunit c (AtpE) is considered an essential target for drug design in multiple bacteria, including mycobacteria where it shares the same pathway with the target of Isoniazid .

• Structural uniqueness: While ATP synthase is conserved, subtle differences between bacterial and mammalian ATP synthase c subunits make it an attractive target for developing selective inhibitors .

• Inhibitor screening approach:

  • In silico screening against homology models of Leptothrix cholodnii AtpE

  • Molecular docking to identify compounds binding with minimum energy

  • Selection of compounds with favorable physicochemical properties

  • Experimental validation through enzyme inhibition assays

Previous studies have identified compounds with binding energies in the range of -8.69 to -8.44 kcal/mol that effectively inhibit bacterial ATP synthase . Similar approaches could identify selective inhibitors for Leptothrix cholodnii, potentially addressing biofilm-associated infections or environmental control where this bacterium is problematic.

How does the sheath structure of Leptothrix cholodnii influence ATP synthase function and organization in the cell membrane?

The unique sheath structure of Leptothrix cholodnii likely has significant implications for membrane protein organization and function:

• Nanofibril-membrane interactions: The nanofibril sheath lowers cell surface hydrophobicity by approximately 60% , which could alter the local membrane environment surrounding ATP synthase complexes.

• Surface charge distribution: Changes in surface hydrophobicity may affect membrane potential and proton gradient maintenance, directly impacting ATP synthase function.

• Spatial organization: The sheath structure may create specialized membrane domains that concentrate ATP synthase complexes in regions optimized for energy generation.

Research approach to investigate this relationship:

• High-resolution cryo-electron microscopy of intact cells to visualize membrane-sheath interfaces

• Fluorescence microscopy with labeled ATP synthase components to track distribution in wild-type vs. sheath-deficient mutants

• Atomic force microscopy to map surface properties and protein organization

• Bioenergetic measurements comparing wild-type and sheath mutants

Understanding this relationship could provide insights into how specialized bacterial structures impact energy metabolism and potentially reveal new targets for disrupting bacterial biofilm formation.

How can researchers overcome challenges in expressing and purifying functional Leptothrix cholodnii ATP synthase subunit c?

Membrane proteins like ATP synthase subunit c present several purification challenges. Here are methodological solutions:

• Challenge: Protein aggregation during expression

  • Solution: Use lower induction temperatures (16-18°C), reduce inducer concentration, and employ specialized expression strains (e.g., C41/C43 for E. coli).

  • Method: Monitor expression using small-scale test expressions at different temperatures (37°C, 25°C, 18°C) with varying IPTG concentrations (0.1-1.0 mM).

• Challenge: Poor solubilization from membranes

  • Solution: Screen multiple detergents individually and in combinations.

  • Method: Test a matrix of detergents (DDM, digitonin, LMNG, CHAPS) at different concentrations with varying solubilization times (1-24 hours).

• Challenge: Loss of structural integrity during purification

  • Solution: Include stabilizing lipids and optimize buffer conditions.

  • Method: Supplement purification buffers with E. coli polar lipid extract (0.1-0.5 mg/mL) and test various pH conditions (pH 6.0-8.0) and salt concentrations (100-500 mM NaCl).

• Challenge: Low yield of functional protein

  • Solution: Optimize reconstitution procedures and verify functional integrity.

  • Method: Use different protein:lipid ratios (1:50 to 1:200) during reconstitution and confirm functionality through proton translocation assays.

These approaches systematically address the key challenges in working with ATP synthase subunit c and increase the likelihood of obtaining functional protein for further studies.

What are the critical factors for successful reconstitution of Leptothrix cholodnii ATP synthase subunit c into liposomes for functional studies?

Successful reconstitution of ATP synthase subunit c requires careful optimization of multiple parameters:

Table 3: Critical Parameters for Liposome Reconstitution of ATP Synthase Subunit c

After reconstitution, functionality should be verified through:

• Proton translocation assays using pH-sensitive fluorophores

• Freeze-fracture electron microscopy to confirm integration and distribution

• ATP synthesis/hydrolysis measurements when combined with other ATP synthase components

Maintaining temperature control (4-25°C) throughout the reconstitution process is essential for preserving protein structure and function.

How can researchers accurately investigate the interaction between Leptothrix cholodnii ATP synthase subunit c and inhibitor compounds?

Investigating interactions between ATP synthase subunit c and potential inhibitors requires a multimodal approach:

• In silico screening:

  • Homology modeling of Leptothrix cholodnii ATP synthase subunit c based on related structures

  • Molecular docking studies to identify binding sites and calculate binding energies

  • Molecular dynamics simulations to assess stability of protein-inhibitor complexes

• Binding assays:

  • Isothermal titration calorimetry (ITC) to determine binding constants and thermodynamic parameters

  • Surface plasmon resonance (SPR) to measure real-time binding kinetics

  • Microscale thermophoresis (MST) for binding studies with minimal protein requirements

• Functional inhibition assessment:

  • ATP synthesis inhibition in reconstituted systems

  • Proton translocation inhibition measured by fluorescence quenching

  • Membrane potential measurements using voltage-sensitive dyes

• Structural confirmation:

  • Hydrogen-deuterium exchange mass spectrometry to map binding interfaces

  • Cryo-EM structure determination of inhibitor-bound complexes

For meaningful results, the inhibitor concentration range should span at least two orders of magnitude around the expected IC50 value, typically from 0.01 to 10 μM for ATP synthase inhibitors . Controls with known inhibitors such as oligomycin should be included for comparison.

How can cryo-electron microscopy be optimized for structural studies of Leptothrix cholodnii ATP synthase?

Cryo-electron microscopy (cryo-EM) offers tremendous potential for structural studies of Leptothrix cholodnii ATP synthase, but requires careful optimization:

• Sample preparation challenges:

  • Protein extraction: Use mild detergents or nanodiscs to maintain native structure

  • Sample concentration: Aim for 2-5 mg/mL final concentration

  • Grid optimization: Test multiple grid types (Quantifoil R1.2/1.3, UltrAuFoil)

  • Vitrification parameters: Optimize blotting time (3-7 seconds) and temperature (4°C)

• Data collection strategy:

  • Exposure settings: Total dose of 40-60 e⁻/Ų distributed across 40-60 frames

  • Defocus range: -0.8 to -2.5 μm

  • Motion correction: Implement per-particle motion correction

  • CTF estimation: Use patch-based CTF estimation for tilted samples

• Image processing workflow:

  • Implement 3D classification to sort heterogeneous conformations

  • Use focused refinement on the c-ring subcomplex

  • Apply symmetry-based averaging appropriate to the c-ring subunit number

This approach can yield structures with resolution in the 2.5-3.5 Å range, sufficient to resolve side-chain conformations and potential inhibitor binding sites in the c-ring assembly.

What roles do specific targeting peptides play in ATP synthase assembly and function in Leptothrix cholodnii?

While the search results don't specifically address targeting peptides in Leptothrix cholodnii, research on mammalian ATP synthase subunit c isoforms provides valuable insights that could be applied to bacterial systems :

• Functional diversity: Different targeting peptides in mammalian ATP synthase subunit c isoforms confer non-redundant functions, affecting protein import and respiratory chain maintenance .

• Research approach for Leptothrix cholodnii:

  • Identify potential signal peptides in the Leptothrix cholodnii atpE gene

  • Create chimeric constructs with signal peptides from different sources

  • Assess localization, assembly efficiency, and functional impact

  • Use RNA interference or CRISPR-based approaches to study specific isoform functions

• Expected outcomes: This research could reveal whether Leptothrix cholodnii utilizes specialized targeting mechanisms for ATP synthase assembly, particularly in the context of its unique sheathed filamentous structure and interface adaptation capabilities .

Understanding these mechanisms could provide insights into bacterial energy metabolism regulation and potentially identify new targets for disrupting bacterial bioenergetics.

How does Leptothrix cholodnii ATP synthase subunit c compare evolutionarily with homologs from other environmental bacteria?

Evolutionary analysis of ATP synthase subunit c provides insights into adaptation mechanisms across bacterial species:

• Sequence conservation analysis:

  • Core functional residues (proton-binding carboxylate, transmembrane helices) show high conservation

  • Surface-exposed residues show greater variability, reflecting adaptation to different membrane environments

  • Specialized adaptations may exist in Leptothrix cholodnii related to its unique ecological niche and sheath structure

• Phylogenetic mapping methods:

  • Maximum likelihood analysis of atpE sequences from diverse bacterial phyla

  • Ancestral sequence reconstruction to identify critical evolutionary transitions

  • Correlation of sequence features with environmental parameters (pH, temperature, oxygen availability)

• Structural comparison approach:

  • Homology modeling based on available c-ring structures

  • Analysis of c-ring size variation and its relationship to proton:ATP stoichiometry

  • Mapping of specialized adaptations to the 3D structure

This comparative approach can reveal how Leptothrix cholodnii ATP synthase has evolved specific adaptations for functioning in its unique ecological niche at oxic-anoxic interfaces where it forms specialized biofilm structures .

How do inhibitor sensitivity profiles differ between Leptothrix cholodnii ATP synthase and homologs from other bacterial species?

Inhibitor sensitivity profiles can vary significantly between bacterial species, providing opportunities for selective targeting:

• Comparative inhibition screening:

  • Test established ATP synthase inhibitors (oligomycin, venturicidin, bedaquiline) against purified enzymes

  • Determine IC50 values and inhibition mechanisms

  • Identify Leptothrix-specific sensitivity or resistance patterns

• Structural basis for differential sensitivity:

  • Map inhibitor binding sites through mutagenesis and structural studies

  • Identify specific residues that confer sensitivity or resistance

  • Design modifications to existing inhibitors for improved selectivity

• Ecological and evolutionary context:

  • Correlate inhibitor sensitivity with environmental pressures

  • Investigate natural products from Leptothrix's habitats as potential inhibitors

  • Explore co-evolution with natural inhibitor producers