Recombinant Bacillus clausii ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c (atpE) in Bacillus clausii?

ATP synthase subunit c (atpE) in Bacillus clausii is a small, hydrophobic protein that forms the c-ring rotor component of the F0 domain in the ATP synthase complex. Structurally, each c-subunit adopts a hairpin-like conformation with two transmembrane helices connected by a small cytoplasmic loop. Multiple c-subunits assemble into a ring structure, with the number varying between 10-15 subunits depending on the organism . The c-ring functions as part of the rotary motor, converting the energy of the proton gradient into mechanical rotation, which drives ATP synthesis in the F1 domain.

In alkaliphilic bacteria like Bacillus clausii, the c-subunit contains specific adaptations that allow the ATP synthase to function efficiently under high pH conditions. Each c-subunit typically contains a conserved proton-binding site with a critical acidic residue (usually aspartate or glutamate) in the middle of the second transmembrane helix that participates in proton translocation. This site is essential for capturing protons from the periplasmic space and releasing them into the cytoplasm as the c-ring rotates against the a-subunit .

How does the c-ring stoichiometry affect ATP synthesis efficiency in alkaliphilic bacteria?

The c-ring stoichiometry (number of c-subunits per ring) directly determines the bioenergetic efficiency of ATP synthesis. The number of c-subunits establishes the H+/ATP ratio, as each 360° rotation of the c-ring results in the synthesis of three ATP molecules.

In alkaliphilic bacteria like Bacillus clausii, the c-ring stoichiometry is a critical parameter because these organisms face bioenergetic challenges due to the low protonmotive force available at high external pH. The research indicates that the number of c-subunits in bacterial ATP synthases can range from 10 to 15, with each organism having a specific number that reflects its bioenergetic adaptations .

What expression systems are optimal for recombinant production of Bacillus clausii ATP synthase subunit c?

When expressing recombinant Bacillus clausii ATP synthase subunit c (atpE), researchers must consider several factors including protein folding, membrane insertion, and potential toxicity. Based on evidence from related ATP synthase studies, the following expression systems have proven effective:

E. coli-based expression systems:
E. coli BL21(DE3) strain has been successfully used for expressing bacterial ATP synthases, including the cotA protein from Bacillus clausii . For membrane proteins like atpE, specialized E. coli strains such as C41(DE3) or C43(DE3) often yield better results as they are designed to tolerate toxic membrane proteins.

Methodological approach:

• Clone the atpE gene into expression vectors with tunable promoters (e.g., pET series with T7 promoter)

• Include a purification tag (His6 or Strep-tag) at the N- or C-terminus with a TEV protease cleavage site

• Transform into expression host and optimize expression conditions

• Use low inducer concentrations (0.1-0.5 mM IPTG) and lower temperatures (18-25°C) to promote proper folding

• For higher yields, consider using auto-induction media

When expressing membrane proteins like atpE, it's critical to optimize membrane insertion and avoid inclusion body formation. Addition of membrane-stabilizing agents such as glycerol (5-10%) or specific detergents at sub-CMC concentrations in the growth media can improve proper membrane insertion.

What are the key considerations for purification of recombinant ATP synthase subunit c proteins?

Purification of hydrophobic membrane proteins like ATP synthase subunit c requires specific approaches to maintain protein stability and function. The following methodological considerations are essential:

Membrane extraction and solubilization:

• Harvest cells and disrupt by sonication or French press in buffer containing protease inhibitors

• Isolate membrane fraction by differential centrifugation (typically 150,000-200,000 × g)

• Solubilize membranes using appropriate detergents - mild detergents like n-dodecyl-β-D-maltoside (DDM), n-decyl-β-D-maltoside (DM), or digitonin are recommended

• Optimize detergent concentration and solubilization time (typically 1-2% detergent, 1-2 hours at 4°C)

Chromatographic purification:

• Perform immobilized metal affinity chromatography (IMAC) for His-tagged proteins

• Follow with size exclusion chromatography to remove aggregates and ensure homogeneity

• Maintain detergent above CMC throughout all purification steps

• Consider using lipid additives (0.1-0.5 mg/ml) to stabilize the protein

Detergent exchange or reconstitution:

• For structural studies, consider detergent exchange to more suitable detergents for crystallization or cryo-EM

• For functional studies, reconstitute into proteoliposomes using E. coli lipids or defined lipid mixtures

The choice of detergent is crucial for maintaining the native structure of the c-subunit. Researchers should evaluate multiple detergents and monitor protein stability using techniques such as size exclusion chromatography and thermal shift assays.

How can site-directed mutagenesis of Bacillus clausii atpE help understand proton translocation mechanisms?

Site-directed mutagenesis of the Bacillus clausii ATP synthase subunit c provides powerful insights into proton translocation mechanisms, especially in the context of alkaliphilic adaptations. The methodological approach should include:

Key residues for mutagenesis:

• The conserved proton-binding aspartate/glutamate in the second transmembrane helix

• Residues potentially involved in alkaliphilic adaptations (e.g., S427Q and V110E substitutions observed in some alkaliphilic species that could repel anions to reduce anion-copper interactions)

• Residues at the interface between adjacent c-subunits

• Residues interacting with the a-subunit during rotation

Systematic mutation strategy:

• Generate conservative substitutions (e.g., Asp→Glu) and more dramatic changes (e.g., Asp→Asn)

• Create chimeric proteins by swapping regions between alkaliphilic and neutrophilic bacterial c-subunits

• Introduce pKa-altering mutations to modify proton affinity

Functional assessment methods:

• ATP synthesis/hydrolysis assays in reconstituted proteoliposomes

• Proton translocation measurements using pH-sensitive fluorescent dyes

• Growth complementation in ATP synthase-deficient strains

• Structural assessment by cryo-EM or X-ray crystallography to evaluate conformational changes

Research on alkaliphilic bacteria has shown that specific adaptations in the ATP synthase are required to overcome the bioenergetic challenges of maintaining ATP synthesis at high external pH. For instance, unlike neutral pH organisms, alkaliphiles cannot rely solely on increasing the transmembrane electrical potential to offset adverse pH gradients . Instead, they may employ specialized mechanisms involving delocalized gradients near the membrane surface and proton transfers via membrane-associated microcircuits between proton pumping complexes and ATP synthases .

What structural techniques are most effective for studying the c-ring of ATP synthase from alkaliphilic bacteria?

Multiple structural biology techniques offer complementary insights into the structure and dynamics of ATP synthase c-rings from alkaliphilic bacteria like Bacillus clausii:

Cryo-electron microscopy (cryo-EM):

• Sample preparation: Purify intact ATP synthase or isolated c-rings in detergent or nanodiscs

• Data collection: Use direct electron detectors and collect motion-corrected image stacks

• Analysis: Apply 3D reconstruction techniques with imposed symmetry based on the c-ring stoichiometry

• Resolution: Can achieve 2.5-3.5 Å resolution for well-behaved samples

• Advantages: Can visualize the entire ATP synthase complex, providing context for c-ring function

X-ray crystallography:

• Sample preparation: Purify c-rings in detergent and screen crystallization conditions

• Crystallization strategies: Use lipidic cubic phase or vapor diffusion methods

• Data collection: Utilize microfocus beamlines for small crystals

• Advantages: Can potentially achieve higher resolution (1.5-2.5 Å) than cryo-EM for small membrane proteins

Solid-state NMR spectroscopy:

• Sample preparation: Express isotopically labeled protein (13C, 15N) and reconstitute into lipid bilayers

• Experiments: Perform distance measurements and determine local structural constraints

• Advantages: Can provide dynamic information in a native-like lipid environment

Comparative analysis of current structural data:
The bacterial c-ring structure has been determined for several species, showing variations in stoichiometry that correlate with their environmental adaptations. For alkaliphilic bacteria, the c-ring architecture might reveal specific features that enable function at high pH. The number of c-subunits observed per rotor varies from 10-15 depending on the organism , with yeast having 10 subunits and spinach chloroplasts having 14 subunits .

What are the common challenges in functional reconstitution of recombinant ATP synthase subunit c?

Functional reconstitution of recombinant ATP synthase subunit c presents several challenges due to its hydrophobic nature and the complexity of assembling a functional c-ring. Researchers should consider the following methodological approaches to address common issues:

Challenge: Insufficient protein incorporation into liposomes
Solution:

• Optimize lipid composition - consider using E. coli total lipids or defined mixtures containing phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin

• Adjust protein-to-lipid ratio (typically start with 1:100 w/w)

• Experiment with different reconstitution methods (detergent removal via dialysis, Bio-Beads, or cyclodextrin)

• Monitor incorporation efficiency using fluorescence quenching or density gradient centrifugation

Challenge: Loss of function during reconstitution
Solution:

• Maintain proper orientation by controlling pH during reconstitution

• Test multiple detergents for initial solubilization

• Include stabilizing agents (glycerol, specific lipids) during the reconstitution process

• Avoid extreme temperatures and pH conditions during preparation

Challenge: Difficulty in measuring c-ring rotation or proton translocation
Solution:

• For rotation assays, site-specifically label the c-ring with fluorescent probes or beads

• For proton translocation, use pH-sensitive fluorescent dyes (ACMA, pyranine)

• Consider co-reconstitution with proton pumps to generate the necessary protonmotive force

• Include appropriate controls to distinguish specific activity from passive leakage

Research on ATP synthases from alkaliphilic bacteria has indicated that specialized "microcircuits" may exist between proton pumping complexes and ATP synthases . When designing reconstitution experiments, consider the potential importance of these spatial relationships for proper function, especially when studying alkaliphile adaptations.

How should researchers analyze ATP synthesis efficiency in alkaliphilic versus neutrophilic ATP synthases?

Comparative analysis of ATP synthesis efficiency between alkaliphilic and neutrophilic ATP synthases requires careful experimental design and analysis:

Experimental setup:

• Reconstitute purified ATP synthases into proteoliposomes under identical conditions

• Establish defined pH gradients across the membrane using buffers of different pH

• Measure ATP synthesis rates at multiple external pH values (ranging from 7.0 to 10.5)

• Simultaneously monitor the magnitude of protonmotive force using voltage-sensitive or pH-sensitive probes

Key parameters to measure:

• ATP synthesis rate (μmol ATP/min/mg protein)

• H+/ATP ratio (determined from initial rates of ATP synthesis and proton uptake)

• Threshold protonmotive force required for ATP synthesis

• pH dependency of catalytic activity (kcat values at different pH)

Data analysis approach:

• Plot ATP synthesis rate versus protonmotive force for both enzyme types

• Calculate and compare thermodynamic efficiency (ATP synthesized per proton translocated)

• Analyze the impact of pH gradient versus membrane potential components separately

Research on alkaliphilic bacteria has shown that they face a bioenergetic conundrum: the protonmotive force at high external pH is theoretically too low to account for the observed ATP synthesis . This is because maintaining a cytoplasmic pH below the external pH creates an energetically adverse pH gradient. Comparative studies could reveal how alkaliphilic ATP synthases overcome this challenge through structural adaptations in the c-subunit and other components .

How can researchers distinguish between structural and functional effects when analyzing ATP synthase c-subunit mutations?

Distinguishing between structural and functional effects of mutations in the ATP synthase c-subunit requires a multi-faceted analytical approach:

Structural assessment:

• Perform circular dichroism (CD) spectroscopy to assess secondary structure integrity

• Use size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to evaluate oligomeric state

• Apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions with altered dynamics

• Assess thermal stability using differential scanning calorimetry (DSC) or thermal shift assays

• If possible, determine high-resolution structures of wild-type and mutant proteins

Functional assessment:

• Measure ATP synthesis and hydrolysis rates at varied pH and ionic conditions

• Analyze proton translocation efficiency using pH-sensitive fluorescent dyes

• Perform rotation assays to directly measure c-ring rotation using single-molecule techniques

• Determine the proton binding affinity through isothermal titration calorimetry (ITC)

Integrated data analysis:

• Correlate structural parameters with functional measurements for each mutation

• Apply principal component analysis to identify patterns across multiple mutations

• Develop structure-function relationship models that can predict the impact of new mutations

• Compare results with molecular dynamics simulations to understand dynamic effects

Example analysis framework:

For alkaliphilic Bacillus clausii, certain mutations might specifically affect the adaptations that enable ATP synthesis at high pH. For example, the S427Q and V110E substitutions observed in some alkaliphiles could affect anion-repulsion mechanisms at the expense of catalytic efficiency, representing an evolutionary trade-off for alkaline adaptation .

What bioinformatic approaches are useful for comparative analysis of atpE sequences across alkaliphilic species?

Comparative bioinformatic analysis of atpE sequences from alkaliphilic and neutrophilic species can reveal evolutionary adaptations that enable ATP synthesis under extreme pH conditions:

Sequence-based analyses:

• Multiple sequence alignment (MSA) using MUSCLE or MAFFT algorithms optimized for membrane proteins

• Phylogenetic tree construction using maximum likelihood methods (RAxML, IQ-TREE)

• Conservation analysis to identify residues universally conserved versus those specific to alkaliphiles

• Positive selection detection using tools like PAML to identify sites under selective pressure

Structure-based analyses:

• Homology modeling based on available c-ring structures

• Electrostatic surface potential calculation to identify changes in charge distribution

• Molecular dynamics simulations to assess differences in proton pathway and protein flexibility

• Protein-protein interaction interface prediction focusing on c-c and c-a interfaces

Integrated bioinformatic workflow:

• Collect and curate atpE sequences from diverse bacteria, annotating pH preference (alkaliphilic, neutrophilic, acidophilic)

• Perform MSA and identify positions showing alkaliphile-specific conservation patterns

• Map these positions onto 3D structural models to evaluate functional significance

• Validate findings through correlation with experimental data on mutants

Key patterns to evaluate:

• Substitutions affecting proton-binding site pKa

• Changes in hydrophobic packing of transmembrane helices

• Adaptations at the c-c subunit interface that could affect c-ring stability or stoichiometry

• Modifications at the stator-rotor interface that might influence proton transfer

Bioinformatic analysis could help identify the specific adaptations that allow alkaliphilic ATP synthases to function efficiently despite the bioenergetic challenges of maintaining ATP synthesis at high external pH. The analysis might reveal how these organisms solve the bioenergetic conundrum where the protonmotive force should theoretically be too low to support observed ATP synthesis rates .

How can recombinant Bacillus clausii ATP synthase subunit c be utilized for bioenergetic research?

Recombinant ATP synthase subunit c from alkaliphilic Bacillus clausii provides a valuable tool for investigating fundamental principles of bioenergetics and energy conversion:

Model system for extreme bioenergetics:

• Use as a platform to study how biological systems overcome thermodynamic constraints

• Investigate the minimum protonmotive force required for ATP synthesis

• Explore the relationship between proton binding affinity and ATP synthesis efficiency

• Examine how membrane composition affects proton transfer and ATP synthase function

Methodological applications:

• Engineer hybrid ATP synthases containing c-subunits from different species to study the contribution of this component to pH adaptation

• Develop reconstituted systems with co-localized respiratory complexes and ATP synthases to study proposed "proton microcircuits"

• Create fluorescently labeled c-rings for single-molecule studies of rotation dynamics

• Design biosensors based on c-ring conformational changes for detecting protonmotive force or pH changes

The research on alkaliphilic bacteria has shown that they employ specialized mechanisms to overcome the bioenergetic challenges of maintaining ATP synthesis at high external pH. These may include delocalized gradients near the membrane surface and proton transfers via membrane-associated microcircuits between proton pumping complexes and ATP synthases . Recombinant Bacillus clausii ATP synthase components can help test these hypotheses experimentally.

What novel insights might be gained from studying the interaction between ATP synthase c-subunits and membrane lipids?

The interaction between ATP synthase c-subunits and membrane lipids represents an important but understudied aspect of ATP synthase function, particularly in extremophiles like alkaliphilic Bacillus clausii:

Key research questions:

• How do lipid-protein interactions contribute to c-ring stability and rotation?

• Do alkaliphilic bacteria employ specific lipid compositions to support ATP synthase function at high pH?

• Can lipid modifications compensate for bioenergetic challenges in alkaline environments?

• How do annular and non-annular lipids differ between alkaliphilic and neutrophilic ATP synthases?

Methodological approaches:

• Lipidomic analysis of native membranes from Bacillus clausii grown at different pH values

• Reconstitution of ATP synthase into nanodiscs with defined lipid compositions

• Site-specific labeling of c-subunits for fluorescence resonance energy transfer (FRET) studies with labeled lipids

• Molecular dynamics simulations to identify specific lipid-binding sites and their impact on protein dynamics

Potential discoveries:

• Identification of lipid binding sites that influence proton access to the c-ring

• Discovery of lipid composition adaptations that support ATP synthesis at high pH

• Understanding of how lipid-protein interactions contribute to the formation of proposed "proton microcircuits"

• Insights into how membrane physical properties (thickness, fluidity, lateral pressure) affect c-ring rotation