Recombinant Bacillus pumilus ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Bacillus pumilus?

ATP synthase subunit c, encoded by the atpE gene in Bacillus pumilus, is a crucial membrane-spanning component of the F-type ATP synthase. In B. pumilus strain SAFR-032, this protein consists of 70 amino acids with the sequence "MNLIAAAIAIGLGALGAGIGNGLIVSKTVEGIARQPEAGRELRTLMFIGVALVEALPIIA VVIAFLAFFG". It forms an oligomeric structure as part of the F0 membrane sector of ATP synthase, playing a vital role in proton translocation across the membrane during ATP synthesis or hydrolysis . The atpE gene in B. pumilus is identified as BPUM_3331 in genomic databases, and the protein functions as a lipid-binding component crucial for energy conversion .

How does the ATP synthase complex function in Bacillus species?

The ATP synthase in Bacillus species is an F-type enzyme composed of two main parts: the membrane-embedded F0 sector and the cytoplasmic F1 sector. This multiprotein complex couples the energy of protons flowing across the cytoplasmic membrane to synthesize ATP from ADP and inorganic phosphate. The F0 sector, which includes subunit c, forms a proton channel through the membrane. When protons flow through this channel, they drive the rotation of the F1 sector, leading to ATP synthesis. In reverse, ATP hydrolysis by F1 can drive proton pumping by F0, generating a proton motive force across the membrane . This bidirectional function allows the enzyme to either synthesize ATP coupled with proton influx or pump protons outward by hydrolyzing ATP, thereby contributing to cellular bioenergetics and pH regulation .

What are the characteristic properties of Bacillus pumilus as a bacterial species?

Bacillus pumilus is a Gram-positive, rod-shaped, endospore-forming bacterium that displays several distinctive properties:

B. pumilus strains typically produce detectable protease and lipase but not amylase, phosphatase, DNase, gelatinase, or chitinase. Regarding antibiotic susceptibility, B. pumilus is generally sensitive to amoxicillin, ciprofloxacin, gentamycin, cotrimaxazole, chloramphenicol, bacitracin, tetracycline, kanamycin, erythromycin, and vancomycin, but resistant to penicillin . Recent studies have also identified pigmented B. pumilus strains that produce C30 carotenoids and riboflavin, which may contribute to their antioxidant properties .

What are the optimal conditions for expressing recombinant B. pumilus atpE protein?

For optimal expression of recombinant B. pumilus ATP synthase subunit c, researchers should consider several factors. The protein's highly hydrophobic nature (as evident from its amino acid sequence) necessitates specialized expression systems. Based on research with similar membrane proteins, E. coli BL21(DE3) or C43(DE3) strains (specifically designed for membrane protein expression) should be used with expression vectors containing strong inducible promoters like T7. Expression should be induced at lower temperatures (16-20°C) to prevent inclusion body formation, with IPTG concentrations between 0.1-0.5 mM. The addition of membrane-stabilizing agents like glycerol (5-10%) to the growth medium can improve yields. For purification, detergent screening is essential, with mild detergents like DDM (n-dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) typically yielding better results for maintaining protein stability and activity .

How should researchers store and handle recombinant B. pumilus atpE protein to maintain its stability?

Recombinant B. pumilus ATP synthase subunit c should be stored in a Tris-based buffer containing 50% glycerol at -20°C, or at -80°C for extended storage periods. To preserve protein integrity, repeated freezing and thawing cycles should be avoided. For ongoing experiments, working aliquots can be stored at 4°C for up to one week . When handling the protein, it's advisable to maintain a reducing environment by including agents like DTT or β-mercaptoethanol in buffers to prevent oxidation of cysteine residues. For functional studies, researchers should consider incorporating the protein into liposomes or nanodiscs to maintain its native conformation and activity. Temperature-controlled environments are crucial during all experimental procedures involving this protein to prevent denaturation.

What methods are most effective for studying the interactions of ATP synthase subunit c with potential inhibitory compounds?

To study interactions between ATP synthase subunit c and potential inhibitory compounds, researchers can employ several complementary approaches:

• Purified protein binding assays: Using techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or microscale thermophoresis (MST) with purified recombinant atpE to measure direct binding kinetics.

• Membrane vesicle assays: Creating inverted membrane vesicles containing ATP synthase to measure ATP hydrolysis activity in the presence of potential inhibitors, as demonstrated in studies with diarylquinolines .

• ATP synthesis/hydrolysis assays: Measuring the effect of compounds on ATP synthesis or hydrolysis using purified ATP synthase or membrane preparations. Inhibition of ATP synthase activity correlates with antibiotic potency, as shown in studies of ATP synthase inhibitors .

• Resistance mutation analysis: Generating resistant mutants through exposure to increasing concentrations of inhibitory compounds, followed by sequencing of the atpE gene to identify mutation sites that confer resistance. This approach has successfully identified the binding sites of compounds like diarylquinolines .

• Structural modeling: Creating homology models of B. pumilus atpE based on crystal structures of related ATP synthases to predict binding sites and interactions with inhibitory compounds .

How does the structure of B. pumilus atpE differ from other bacterial species, and what implications might these differences have for antimicrobial targeting?

Structural comparison of B. pumilus ATP synthase subunit c with other bacterial species reveals both conserved and variable regions that have significant implications for antimicrobial targeting. The amino acid sequence of B. pumilus atpE (MNLIAAAIAIGLGALGAGIGNGLIVSKTVEGIARQPEAGRELRTLMFIGVALVEALPIIA VVIAFLAFFG) shows conservation of key functional residues involved in proton translocation, particularly the essential ion-binding glutamate residue (equivalent to Glu54 in other species) .

The specific structure of atpE in Bacillus species has been implicated in narrow spectrum activity of certain antibiotics. For example, diarylquinolines show specific activity against Bacillales, suggesting that conserved sequences in ATP synthase subunit c across this order may provide targeting opportunities . These structural differences could be exploited for the development of narrow-spectrum antibiotics that specifically target Bacillus species while sparing other beneficial bacteria.

What role does ATP synthase subunit c play in bacterial resistance to antimicrobial compounds?

ATP synthase subunit c plays a crucial role in bacterial resistance to certain antimicrobial compounds through several mechanisms:

• Target-site mutations: Mutations in the atpE gene can significantly alter the binding affinity of antimicrobial compounds. Studies with ATP synthase inhibitors have identified specific mutations (e.g., changes in Ala17, Gly18, Ser26, and Phe47 in S. aureus) that confer high-level resistance . These mutations typically occur near essential ion-binding sites (like Glu54), suggesting that resistance mutations must maintain protein functionality while preventing drug binding.

• Energy metabolism adaptation: Bacteria with mutations in atpE often show altered ATP metabolism. Interestingly, ATP synthase mutants resistant to compounds like tomatidine derivatives have been shown to have further reduced ATP production compared to parent strains, indicating metabolic compensation mechanisms .

• Frequency of resistance: The mutation frequency for ATP synthase inhibitors is relatively low (approximately 10^-7 to 10^-6 at 5× to 50× MIC for S. pneumoniae), making resistance development less common than for some other antibiotics .

• Cross-resistance patterns: Mutations in atpE can confer cross-resistance to multiple ATP synthase inhibitors, but the level of resistance varies. For example, resistance to tomatidine can provide varying levels of cross-resistance to its derivatives .

Understanding these resistance mechanisms is crucial for developing combination therapies or novel derivatives that can overcome resistance. For example, FC04-100, a derivative of tomatidine, prevents high-level resistance development in some bacterial strains and limits resistance in small-colony variants .

How does the ATP synthase complex interact with other cellular components in B. pumilus?

The ATP synthase complex in Bacillus species forms intricate interactions with various cellular components, creating a sophisticated energy production network:

• Interaction with mitochondrial calcium uniporter (MCU) homologs: Research has shown that ATP synthase subunit c interacts with MCU components in other organisms, suggesting potential regulatory connections between calcium signaling and energy production. This interaction may form a "megacomplex" that couples ADP and Pi transport with ATP synthesis in a process stimulated by calcium .

• Association with ATP transport proteins: The ATP operon in Bacillus species contains additional genes like atpZ and atpI, which encode membrane proteins involved in magnesium and calcium transport. These proteins may facilitate the provision of Mg^2+, which is required by ATP synthase, and support charge compensation when the enzyme functions in the hydrolytic direction .

• Integration with respiratory chain components: ATP synthase functionally couples with respiratory chain complexes to harness the proton gradient generated during respiration. This coupling is essential for efficient energy production, especially under aerobic conditions .

• Role in biofilm formation: The ATP synthase activity may be linked to biofilm formation through energy provision for extracellular matrix production. In Bacillus species, glycosyltransferases involved in biofilm formation, such as cellulose synthase GT2, have been identified alongside ATP synthase components in genomic studies .

• Participation in large protein complexes: Blue native PAGE analysis has revealed that ATP synthase can exist in large protein complexes with molecular weights of approximately 900 kDa, suggesting association with other membrane proteins or multimerization .

Understanding these interactions provides insights into the integrated nature of energy metabolism in B. pumilus and may reveal new targets for antimicrobial development or biotechnological applications.

How can recombinant B. pumilus atpE be utilized in screening for novel antimicrobial compounds?

Recombinant B. pumilus ATP synthase subunit c can serve as a valuable tool for screening novel antimicrobial compounds through several sophisticated approaches:

• Target-based high-throughput screening: Purified recombinant atpE protein can be incorporated into liposomes or nanodiscs for fluorescence-based binding assays to screen large compound libraries. Changes in intrinsic fluorescence or using fluorescent probes can detect binding events.

• ATP synthesis/hydrolysis inhibition assays: By reconstituting functional ATP synthase complexes containing B. pumilus atpE, researchers can develop assays that measure ATP production or hydrolysis in the presence of test compounds. For ATP hydrolysis assays, the release of inorganic phosphate can be monitored colorimetrically, while ATP synthesis can be measured using luciferase-based assays .

• Membrane potential assays: Since ATP synthase activity is linked to proton translocation, compounds that target atpE can alter membrane potential. Fluorescent dyes like DiSC3(5) that respond to membrane potential changes can be utilized in bacterial cells or membrane vesicles to identify compounds that disrupt ATP synthase function .

• Resistance mutation mapping: By creating a library of atpE variants with systematic mutations and testing their susceptibility to candidate compounds, researchers can map the binding sites and structure-activity relationships of potential antimicrobials. This approach has been successful in identifying the binding mode of diarylquinolines to ATP synthase .

• Selectivity screening: Comparative testing against human mitochondrial ATP synthase components can identify compounds with selectivity for bacterial ATP synthase. The selectivity index (inhibition of bacterial ATP synthase versus mitochondrial ATP production) should be high (>10^5) for promising antimicrobial candidates .

What are the potential roles of B. pumilus ATP synthase in biotechnological applications beyond antimicrobial development?

B. pumilus ATP synthase has several promising biotechnological applications beyond antimicrobial development:

• Bioenergy production: The high efficiency of ATP synthase in converting proton gradient energy to chemical energy makes it valuable for bioenergy applications. Engineered systems incorporating B. pumilus ATP synthase components could potentially be used for ATP production in bioreactors or for coupling to artificial photosynthetic systems.

• Biosensors: The sensitivity of ATP synthase to various environmental factors (pH, ion concentrations, inhibitors) makes it suitable for biosensor development. For instance, B. pumilus atpE-based sensors could detect contamination with specific toxins or antimicrobials that target ATP synthase.

• Vaccine development: B. pumilus spores expressing recombinant antigens, potentially including modified atpE, have shown promise as vaccine delivery systems. These spores can survive passage through the stomach, germinate in the gut, and form biofilms that present antigens to stimulate immune responses .

• Drug delivery systems: The understanding of ATP synthase structure and function can inspire the design of novel nanostructures for drug delivery, particularly for targeting bacterial infections.

• Agricultural applications: B. pumilus strains have been identified as plant growth-promoting bacteria (PGPB), with ATP synthase function potentially contributing to their stress resistance and survival in the soil. Research shows that B. pumilus can produce phytohormones (gibberellins, indole-3-acetic acid), solubilize phosphate, and fix atmospheric nitrogen, making them valuable for sustainable agriculture .

What are the current challenges in studying B. pumilus ATP synthase and how might they be overcome?

Researchers face several significant challenges when studying B. pumilus ATP synthase:

• Protein expression and purification difficulties: The hydrophobic nature of atpE and other membrane components of ATP synthase makes expression and purification challenging. This can be addressed by:

  • Using specialized expression systems designed for membrane proteins

  • Employing fusion tags that enhance solubility

  • Developing optimized detergent screening protocols

  • Exploring alternative systems like cell-free expression

• Structural characterization limitations: Obtaining high-resolution structures of membrane proteins like atpE is notoriously difficult. Emerging approaches include:

  • Cryo-electron microscopy (cryo-EM) for structure determination without crystallization

  • Solid-state NMR for studying membrane proteins in native-like environments

  • Molecular dynamics simulations to predict structural dynamics

• Functional reconstitution challenges: Reconstituting fully functional ATP synthase complexes in vitro is complex. Innovative approaches include:

  • Developing improved proteoliposome systems with controlled lipid composition

  • Using nanodiscs or other membrane mimetics to maintain protein activity

  • Employing microfluidic systems for high-throughput functional analysis

• Species-specific differences: Extrapolating findings from model organisms to B. pumilus can be problematic due to species-specific variations. Researchers should:

  • Conduct comparative genomic and proteomic analyses across Bacillus species

  • Develop B. pumilus-specific genetic tools for in vivo studies

  • Establish standardized assays that account for species differences

• Integration of in vivo and in vitro findings: Connecting biochemical observations to physiological relevance remains challenging. Approaches to bridge this gap include:

  • Developing B. pumilus-specific genetic tools for targeted mutations

  • Using systems biology approaches to model ATP synthase within cellular networks

  • Employing advanced imaging techniques to study ATP synthase in living cells

How might emerging technologies advance our understanding of ATP synthase function and regulation in B. pumilus?

Emerging technologies offer exciting opportunities to deepen our understanding of ATP synthase in B. pumilus:

• Cryo-electron microscopy advancements: Recent breakthroughs in cryo-EM enable visualization of membrane protein complexes at near-atomic resolution. Applied to B. pumilus ATP synthase, this could reveal:

  • Detailed structural organization of the F0F1 complex

  • Conformational changes during catalysis

  • Species-specific structural features relevant to antimicrobial targeting

• Single-molecule techniques: Technologies like single-molecule FRET and high-speed AFM can track conformational dynamics of ATP synthase in real-time, potentially revealing:

  • Rotational mechanics of the enzyme

  • Coupling mechanisms between proton translocation and ATP synthesis

  • Effects of inhibitors on enzyme dynamics

• CRISPR-Cas9 genome editing: Advanced genetic tools enable precise manipulation of the ATP synthase operon in B. pumilus to:

  • Create site-specific mutations to study structure-function relationships

  • Generate reporter constructs to monitor expression and localization

  • Develop conditional knockdowns to assess physiological roles

• Synthetic biology approaches: Synthetic reconstructions of ATP synthase components could enable:

  • Creation of minimal functional units to define essential components

  • Design of hybrid systems with components from different species

  • Development of engineered ATP synthases with novel properties

• Multi-omics integration: Combining genomics, transcriptomics, proteomics, and metabolomics can provide a systems-level understanding of:

  • How ATP synthase expression responds to environmental conditions

  • Regulatory networks controlling ATP synthase function

  • Metabolic consequences of ATP synthase modulation

• Microfluidics and organ-on-chip platforms: These technologies could enable:

  • Real-time monitoring of ATP synthase activity in response to changing conditions

  • High-throughput screening of environmental factors affecting enzyme function

  • Modeling of host-pathogen interactions involving energy metabolism

What are the potential implications of ATP synthase research for understanding bacterial adaptation and evolution?

Research on ATP synthase in B. pumilus has profound implications for understanding bacterial adaptation and evolution:

• Evolutionary conservation and divergence: The ATP synthase complex represents one of the most ancient and conserved energy-converting enzymes. Comparative analysis of B. pumilus atpE with other species reveals:

  • Highly conserved functional residues essential for proton translocation

  • Species-specific variations that may reflect adaptation to different ecological niches

  • Insights into the co-evolution of energy metabolism and other cellular processes

• Adaptation to environmental stresses: B. pumilus is known for its resistance to extreme conditions, including UV radiation, oxidative stress, and desiccation. ATP synthase may play a critical role in these adaptations through:

  • Maintaining energy homeostasis under stress conditions

  • Contributing to pH regulation via proton pumping

  • Supporting spore formation and germination processes

• Antimicrobial resistance mechanisms: Studies of ATP synthase inhibitors reveal how bacteria can develop resistance through specific mutations in atpE. This provides insights into:

  • Constraint-based evolution where functional requirements limit mutation options

  • Trade-offs between resistance and fitness (e.g., reduced ATP production in resistant mutants)

  • Predictive models for resistance development to new ATP synthase-targeting compounds

• Horizontal gene transfer considerations: The ATP synthase operon structure and organization across bacterial species offer perspectives on:

  • The role of horizontal gene transfer in shaping energy metabolism

  • Acquisition of additional genes like atpZ that may provide functional advantages

  • Co-evolution of ATP synthase with respiratory chain components

• Metabolic plasticity: Research reveals how bacteria can adapt their energy metabolism pathways in response to ATP synthase inhibition or mutation:

  • Alternative ATP generation pathways that become activated

  • Metabolic remodeling that occurs in persistent or slow-growing bacteria

  • Connections between energy metabolism and virulence or stress responses

Understanding these evolutionary aspects not only provides fundamental biological insights but also informs strategies for antimicrobial development that consider evolutionary constraints and potential resistance mechanisms.

What is the recommended protocol for purifying recombinant B. pumilus ATP synthase subunit c for structural studies?

Protocol for Purification of Recombinant B. pumilus ATP Synthase Subunit c for Structural Studies

Materials Required:

• pET-based expression vector containing B. pumilus atpE gene

• E. coli C43(DE3) cells (specialized for membrane protein expression)

• Terrific Broth (TB) medium with appropriate antibiotics

• IPTG (isopropyl β-D-1-thiogalactopyranoside)

• Phosphate-buffered saline (PBS)

• DDM (n-dodecyl β-D-maltoside) or LMNG (lauryl maltose neopentyl glycol) detergent

• Ni-NTA agarose resin

• Size exclusion chromatography (SEC) column

• Tris-based buffer with 50% glycerol for storage

Procedure:

• Transformation and Expression:

  • Transform E. coli C43(DE3) cells with the expression vector containing B. pumilus atpE

  • Grow a starter culture in LB medium with appropriate antibiotics at 37°C overnight

  • Inoculate TB medium (1:100 dilution) and grow at 37°C until OD600 reaches 0.6-0.8

  • Cool the culture to 18°C and induce with 0.5 mM IPTG

  • Continue growth at 18°C for 16-18 hours

• Cell Harvest and Membrane Preparation:

  • Harvest cells by centrifugation (6,000 × g, 15 min, 4°C)

  • Resuspend in PBS buffer containing 1 mM PMSF and 5 mM β-mercaptoethanol

  • Lyse cells using a high-pressure homogenizer (3 passes at 15,000 psi)

  • Remove unbroken cells and debris by centrifugation (20,000 × g, 30 min, 4°C)

  • Collect membranes by ultracentrifugation of the supernatant (150,000 × g, 1 hour, 4°C)

• Protein Solubilization:

  • Resuspend membrane pellet in solubilization buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 5% glycerol)

  • Add DDM to a final concentration of 1% (w/v) or LMNG to 1% (w/v)

  • Incubate with gentle rotation at 4°C for 2 hours

  • Remove insoluble material by ultracentrifugation (150,000 × g, 30 min, 4°C)

• Affinity Purification:

  • Equilibrate Ni-NTA resin with binding buffer (20 mM Tris-HCl pH 8.0, 300 mM NaCl, 0.05% DDM or 0.01% LMNG, 5% glycerol, 20 mM imidazole)

  • Incubate the solubilized protein with the resin for 2 hours at 4°C

  • Wash the resin extensively with wash buffer (binding buffer with 50 mM imidazole)

  • Elute the protein with elution buffer (binding buffer with 300 mM imidazole)

• Size Exclusion Chromatography:

  • Concentrate the eluted protein using a 10 kDa MWCO concentrator

  • Load the concentrated protein onto a Superdex 200 column equilibrated with SEC buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% DDM or 0.01% LMNG, 5% glycerol)

  • Collect fractions containing the purified protein

• Quality Control:

  • Assess protein purity by SDS-PAGE (use special conditions for membrane proteins: sample buffer containing 2% SDS, no boiling)

  • Verify protein identity by Western blotting and/or mass spectrometry

  • Check for proper folding using circular dichroism spectroscopy

• Storage:

  • Concentrate the purified protein to 1-5 mg/mL

  • Add glycerol to a final concentration of 50%

  • Aliquot and flash-freeze in liquid nitrogen

  • Store at -80°C for long-term storage

Critical Considerations:

• All buffers should be ice-cold and contain protease inhibitors

• Maintain detergent concentrations above critical micelle concentration throughout purification

• For structural studies (X-ray crystallography or cryo-EM), consider removing the affinity tag by proteolytic cleavage after the affinity purification step

• For NMR studies, consider expression in minimal media with appropriate isotope labeling

How can researchers effectively perform mutagenesis studies to investigate structure-function relationships in B. pumilus atpE?

Comprehensive Protocol for Mutagenesis Studies of B. pumilus atpE

Strategy Overview:
To investigate structure-function relationships in B. pumilus ATP synthase subunit c, a systematic approach combining site-directed mutagenesis with functional assays is recommended. Based on methods used in similar studies , the following protocol outlines key steps and considerations.

Materials Required:

• Plasmid containing B. pumilus atpE gene

• Mutagenesis primers for targeted mutations

• High-fidelity DNA polymerase (e.g., Phusion or Q5)

• DpnI restriction enzyme

• Competent E. coli cells for cloning

• Expression host (E. coli C43(DE3) or Bacillus expression system)

• Materials for protein purification and functional assays

Procedure:

• Selection of Mutation Sites:

  • Based on sequence alignment with homologous proteins where function is better characterized

  • Focus on conserved residues such as the ion-binding glutamate (equivalent to Glu54 in S. aureus)

  • Target residues implicated in antimicrobial resistance (e.g., equivalents to Ala17, Gly18, Ser26, and Phe47 in S. aureus)

  • Include transmembrane residues that might be involved in proton translocation

• Primer Design for Site-Directed Mutagenesis:

  • Design mutagenic primers (25-35 nucleotides) with the desired mutation centrally located

  • Ensure primers have a Tm ≥78°C (for QuikChange-type protocols)

  • Include silent mutations to create diagnostic restriction sites for screening

  • Verify primer specificity using sequence analysis tools

• PCR-Based Mutagenesis:

  • Perform PCR using high-fidelity polymerase with the following conditions:

    • Initial denaturation: 98°C for 30 seconds

    • 16-18 cycles of: 98°C for 10 seconds, 55-68°C for 30 seconds, 72°C for 30 seconds/kb

    • Final extension: 72°C for 10 minutes

  • Digest parental plasmid with DpnI (37°C for 1 hour)

  • Transform into competent E. coli cells

  • Screen colonies using diagnostic restriction digestion or colony PCR

  • Verify mutations by DNA sequencing

• Expression of Mutant Proteins:

  • Transform expression plasmids into E. coli C43(DE3) or Bacillus expression host

  • Optimize expression conditions for each mutant (temperature, inducer concentration, duration)

  • Establish control expressions of wild-type protein in parallel

  • Extract and purify proteins using the protocol outlined in FAQ 6.1

• Functional Characterization:

  • ATP Synthesis/Hydrolysis Assays:

    • Reconstitute purified proteins into liposomes

    • Measure ATP synthesis driven by artificial proton gradient

    • Assess ATP hydrolysis using phosphate release assays

  • Proton Translocation Assays:

    • Prepare protein-reconstituted liposomes with pH-sensitive fluorescent dyes

    • Monitor proton translocation in response to ATP addition

    • Compare kinetics between wild-type and mutant proteins

  • Binding Studies with ATP Synthase Inhibitors:

    • Perform binding assays using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC)

    • Compare binding affinities of inhibitors to wild-type and mutant proteins

    • Correlate binding data with functional effects

• Structural Analysis:

  • Create structural models of wild-type and mutant proteins using homology modeling

  • Analyze potential structural changes using molecular dynamics simulations

  • For key mutants, consider structural determination using X-ray crystallography or cryo-EM

• Complementation Studies in Bacterial Systems:

  • Introduce mutant atpE genes into atpE-deficient bacterial strains

  • Assess growth phenotypes under various conditions (pH, temperature, stress)

  • Measure ATP synthesis in membrane vesicles prepared from complemented strains

Data Analysis and Interpretation:

• Compare biochemical parameters (Km, Vmax, binding constants) between wild-type and mutant proteins

• Assess structure-function correlations based on the mutational effects

• Identify critical residues for different aspects of ATP synthase function

• Develop a mechanistic model for B. pumilus ATP synthase operation

Potential Challenges and Solutions:

• Some mutations may destabilize the protein: Screen multiple expression conditions or use fusion partners to improve stability

• Functional assays may be difficult to standardize: Include appropriate controls and perform statistical analyses on multiple independent experiments

• Interpretation of complex phenotypes: Use combinatorial mutations and compare with homologous systems where more data is available

What is the most effective methodology for studying the interaction between B. pumilus ATP synthase and the bacterial membrane environment?

Methodology for Studying B. pumilus ATP Synthase-Membrane Interactions

Understanding the interaction between B. pumilus ATP synthase and its membrane environment requires specialized techniques that preserve native interactions while allowing detailed analysis. The following comprehensive methodology integrates multiple approaches:

Preparation of Native Membrane Environments

A. Inverted Membrane Vesicles (IMVs):

• Harvest B. pumilus cells at mid-logarithmic phase

• Disrupt cells using French press or sonication

• Collect membrane vesicles by differential centrifugation

• Verify inversion using enzyme accessibility assays

• Measure ATP synthesis/hydrolysis activities using established protocols

B. Lipid Extraction and Characterization:

• Extract total lipids from B. pumilus membranes using Bligh-Dyer method

• Analyze lipid composition by thin-layer chromatography and mass spectrometry

• Determine lipid/protein ratio in native membranes

• Identify specific lipids associated with purified ATP synthase complexes

Reconstitution Systems

A. Proteoliposome Preparation:

• Reconstitute purified ATP synthase into liposomes with defined lipid composition

• Create liposomes mimicking B. pumilus membrane lipid composition

• Compare function in native-like versus synthetic lipid environments

• Assess the impact of specific lipids (cardiolipin, phosphatidylglycerol) on enzyme activity

B. Nanodiscs Assembly:

• Prepare membrane scaffold protein (MSP)

• Reconstitute ATP synthase into nanodiscs with controlled lipid composition

• Verify proper assembly using size-exclusion chromatography and electron microscopy

• Compare functional properties in different lipid environments

Biophysical Analysis of Membrane Interactions

A. Fluorescence Spectroscopy:

• Label ATP synthase components with environmentally sensitive probes

• Monitor conformational changes in different membrane environments

• Use FRET to measure distances between subunits in the membrane

• Assess lipid fluidity effects on protein dynamics

B. Solid-State NMR Spectroscopy:

• Prepare isotopically labeled ATP synthase components

• Study protein-lipid interactions at atomic resolution

• Measure distances between protein segments and lipid headgroups

• Identify specific lipid binding sites

C. Atomic Force Microscopy (AFM):

• Image ATP synthase in supported lipid bilayers

• Measure mechanical properties of protein-membrane complexes

• Observe conformational changes under different conditions

• Assess oligomeric state and organization in the membrane

Functional Analysis in Membrane Context

A. Proton Translocation Assays:

• Monitor pH changes using fluorescent probes (ACMA, pyranine)

• Measure proton pumping activity in response to ATP hydrolysis

• Assess the effect of membrane composition on proton translocation efficiency

• Compare kinetics in different reconstitution systems

B. Membrane Potential Measurements:

• Use voltage-sensitive dyes (DiSC3(5), Oxonol VI) to monitor membrane potential

• Assess the impact of ATP synthase activity on membrane potential

• Measure the effect of inhibitors on membrane energization

• Correlate membrane potential changes with ATP synthesis/hydrolysis

Advanced Imaging Techniques

A. Cryo-Electron Microscopy (Cryo-EM):

• Visualize ATP synthase in membrane environments

• Determine structural organization in lipid bilayers

• Assess the impact of lipid composition on protein conformation

• Analyze supramolecular assemblies and potential interactions with other membrane proteins

B. Super-Resolution Microscopy:

• Label specific ATP synthase subunits with fluorescent probes

• Observe distribution and dynamics in native membranes

• Measure diffusion coefficients in different membrane environments

• Identify potential membrane microdomains associated with ATP synthase

Computational Approaches

A. Molecular Dynamics Simulations:

• Build models of B. pumilus ATP synthase in lipid bilayers

• Simulate protein-lipid interactions over extended timeframes

• Analyze lipid binding sites and their impact on protein structure

• Assess the effect of membrane thickness and curvature on protein function

B. Coarse-Grained Simulations:

• Model larger-scale organization and dynamics

• Assess protein clustering or segregation in complex membranes

• Predict the impact of membrane composition on protein function

• Identify potential lipid-mediated protein-protein interactions

Data Integration and Analysis:

• Correlate structural data with functional measurements

• Compare results across different experimental platforms

• Develop comprehensive models of ATP synthase-membrane interactions

• Identify critical lipid-protein interactions for optimal function