Recombinant Pseudomonas fluorescens ATP synthase subunit c (atpE)

What is the structure and function of ATP synthase subunit c in Pseudomonas fluorescens?

ATP synthase subunit c (atpE) in Pseudomonas fluorescens is a small hydrophobic protein that forms the c-ring in the F₀ domain of ATP synthase. This c-ring functions as a proton channel and participates in the rotary mechanism that drives ATP synthesis. The protein typically has a hairpin structure with two transmembrane α-helices connected by a polar loop. Similar to its counterpart in Pseudomonas aeruginosa, the protein likely forms an oligomeric ring structure embedded in the membrane, with each c-subunit containing a proton-binding site (typically an aspartate or glutamate residue) essential for proton translocation . The structure-function relationship is critical for understanding energy transduction mechanisms in bacterial systems and has implications for both basic research and biotechnological applications.

How does recombinant P. fluorescens ATP synthase subunit c differ from native protein?

Recombinant P. fluorescens ATP synthase subunit c typically includes modifications that facilitate purification and experimental manipulation. These modifications commonly include:

When working with recombinant versions, researchers should verify that the added elements don't significantly alter the protein's structural integrity or functional properties. Expression in heterologous systems like E. coli (similar to P. aeruginosa ATP synthase subunit a) can affect protein folding and assembly characteristics .

What are the recommended storage conditions for recombinant P. fluorescens atpE protein?

For optimal stability and activity maintenance of recombinant P. fluorescens atpE protein, implement the following storage protocol:

• Store lyophilized protein at -20°C to -80°C for long-term storage

• After reconstitution, store working aliquots at 4°C for up to one week

• For reconstituted protein storage beyond one week, add glycerol to a final concentration of 50% and store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles as they significantly decrease protein stability and activity

• Use Tris/PBS-based buffer with 6% trehalose at pH 8.0 for storage buffer composition

These conditions are extrapolated from protocols for similar ATP synthase components and should maintain protein integrity . For membrane proteins like atpE, consider adding appropriate detergents to prevent aggregation during storage.

What expression systems are most effective for producing recombinant P. fluorescens atpE protein?

The selection of an expression system for recombinant P. fluorescens atpE requires careful consideration of multiple factors that influence protein yield, folding, and functionality:

For most research applications, E. coli expression systems represent the optimal balance between yield and functionality, similar to the approach used for P. aeruginosa ATP synthase components . When expressing in E. coli, fusion to an N-terminal His-tag facilitates purification while minimally impacting protein structure and function. For membrane proteins like atpE, specialized E. coli strains (C41/C43) often provide better results by accommodating the potential toxicity of membrane protein overexpression.

How can I optimize the purification protocol for recombinant P. fluorescens atpE?

Optimizing purification of recombinant P. fluorescens atpE requires a multi-step approach tailored to this highly hydrophobic membrane protein:

• Membrane Solubilization:

  • Use mild detergents like n-dodecyl-β-D-maltoside (DDM) at 1-2% for initial solubilization

  • Incubate solubilization mixture at 4°C for 1-2 hours with gentle rotation

  • Centrifuge at 100,000 × g for 1 hour to remove insoluble material

• Affinity Chromatography:

  • For His-tagged constructs, use Ni-NTA resin with imidazole gradient elution (10-500 mM)

  • Maintain detergent concentration at 0.1% throughout purification

  • Include 10% glycerol in all buffers to enhance protein stability

• Size Exclusion Chromatography:

  • Apply sample to Superdex 200 column for final purification

  • Use buffer containing 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.05% DDM

• Quality Control:

  • Verify purity by SDS-PAGE (should exceed 90%)

  • Confirm identity by Western blot or mass spectrometry

  • Assess oligomeric state by native PAGE or analytical ultracentrifugation

This protocol can be adapted based on specific research requirements and equipment availability. For functional studies, consider incorporating additional steps to reconstitute the protein into liposomes or nanodiscs.

What analytical methods are most informative for characterizing recombinant P. fluorescens atpE structure?

Comprehensive structural characterization of recombinant P. fluorescens atpE requires a multi-technique approach to address different structural aspects:

For initial characterization, combining CD spectroscopy and FTIR provides accessible information about secondary structure content. For higher-resolution analysis, cryo-EM has emerged as particularly valuable for membrane protein complexes like ATP synthase components. When designing these experiments, ensure that the protein is maintained in a native-like environment, typically using detergent micelles, nanodiscs, or liposomes to preserve structural integrity.

How can I assess the functional integrity of recombinant P. fluorescens atpE?

Verifying the functional integrity of recombinant P. fluorescens atpE involves multiple complementary approaches since this protein functions as part of the larger ATP synthase complex:

• Proton Translocation Assays:

  • Reconstitute purified atpE into liposomes containing pH-sensitive fluorescent dyes (ACMA or pyranine)

  • Establish a pH gradient across the membrane and monitor fluorescence changes

  • Quantify proton transport rates under varying conditions (pH, membrane potential)

• Oligomeric Assembly Assessment:

  • Use blue native PAGE to verify formation of c-ring oligomers

  • Apply cross-linking approaches with MS analysis to confirm appropriate subunit interactions

  • Employ analytical ultracentrifugation to determine oligomeric state

• Binding Studies with Partner Subunits:

  • Perform co-immunoprecipitation with other ATP synthase components

  • Use surface plasmon resonance to quantify binding affinities with subunits a and b

  • Apply microscale thermophoresis for interaction analysis in detergent environments

• Reconstitution into ATP Synthase Complex:

  • Combine with other purified subunits to assess complex formation

  • Measure ATP synthesis/hydrolysis activity of reconstituted complexes

  • Compare kinetic parameters with those of native enzyme complexes

When conducting these analyses, always include appropriate controls such as well-characterized ATP synthase subunits from model organisms and known inhibitors of ATP synthase activity.

What methods can detect protein-protein interactions between atpE and other ATP synthase components?

Detecting and characterizing protein-protein interactions between atpE and other ATP synthase components requires specialized techniques suitable for membrane protein complexes:

For membrane proteins like ATP synthase components, chemical cross-linking followed by mass spectrometry analysis provides particularly valuable insights, as it can identify specific residues involved in subunit interfaces. When combined with computational modeling, these approaches can generate structural models of the assembled complex. Similar approaches have been successfully applied to study RNA-protein interactions in Pseudomonas species .

How do mutations in conserved residues of P. fluorescens atpE affect ATP synthase function?

Mutations in conserved residues of P. fluorescens atpE can profoundly impact ATP synthase function through several mechanisms:

When designing mutation studies, consider using site-directed mutagenesis to systematically alter conserved residues and assess the impact on:

• Protein expression and stability

• Oligomeric assembly

• Proton translocation efficiency

• ATP synthesis/hydrolysis coupling

These mutation studies not only provide insights into structure-function relationships but also help identify potential antimicrobial target sites, as ATP synthase is essential for bacterial energy metabolism.

How do environmental factors influence the stability and function of recombinant P. fluorescens atpE?

Environmental factors significantly impact the stability and function of recombinant P. fluorescens atpE, with important implications for experimental design and data interpretation:

Research indicates that P. fluorescens ATP synthase displays adaptations to environmental conditions that differ from those of other bacterial species. When designing experiments, carefully control and report these environmental parameters to ensure reproducibility. For long-term storage, lyophilization with appropriate protectants (e.g., trehalose) offers superior stability compared to liquid formulations .

What are the critical differences between ATP synthase subunit c from P. fluorescens and other bacterial species?

Comparative analysis reveals significant differences between ATP synthase subunit c from P. fluorescens and other bacterial species that influence both structure and function:

These differences reflect evolutionary adaptations to different ecological niches and energy requirements. When designing experiments with P. fluorescens atpE, researchers should consider these species-specific characteristics rather than directly applying protocols optimized for E. coli or other model organisms. The structural and functional differences may also explain variations in antimicrobial susceptibility between these bacterial species.

How can molecular dynamics simulations enhance our understanding of P. fluorescens atpE function?

Molecular dynamics (MD) simulations provide powerful insights into the dynamic behavior of P. fluorescens atpE at atomic resolution, complementing experimental approaches:

• Proton Translocation Mechanism:

  • Simulations can reveal the detailed pathway of proton movement through the c-ring

  • Identify water molecules and amino acid side chains involved in proton transfer

  • Calculate energy barriers for proton translocation steps

• Lipid-Protein Interactions:

  • Model interactions between atpE and specific membrane lipids

  • Identify lipid binding sites that may stabilize the c-ring structure

  • Assess the impact of membrane composition on protein dynamics

• Conformational Dynamics:

  • Track structural changes during rotation and proton binding/release

  • Identify flexible regions and conformational states not captured in static structures

  • Calculate free energy profiles for different functional states

• Subunit Interface Analysis:

  • Model interactions between c-subunits in the c-ring assembly

  • Characterize the interface between c-ring and a-subunit

  • Identify key residues that determine specificity of subunit interactions

When conducting MD simulations, ensure that the starting structure accurately represents the P. fluorescens atpE protein, as structural details can significantly impact simulation results. Similar computational approaches have been successfully applied to study protein-RNA interactions in Pseudomonas species .

What are common challenges in expressing and purifying functional P. fluorescens atpE?

Researchers frequently encounter several challenges when working with recombinant P. fluorescens atpE, each requiring specific troubleshooting approaches:

For membrane proteins like atpE, maintaining a native-like environment throughout expression and purification is crucial. Consider incorporating a reconstitution step into liposomes or nanodiscs immediately after purification to stabilize the protein. Similar approaches have been effective for other membrane proteins from Pseudomonas species, where maintaining proper folding during purification is critical for experimental success .

How can I distinguish between functional and non-functional forms of recombinant P. fluorescens atpE?

Distinguishing between functional and non-functional forms of recombinant P. fluorescens atpE requires a multi-parameter assessment approach:

• Structural Integrity Assessment:

  • CD spectroscopy to verify secondary structure content (should show predominant α-helical signature)

  • Tryptophan fluorescence spectroscopy to assess tertiary structure (blue shift indicates proper folding)

  • SEC-MALS to confirm appropriate oligomeric state

• Functional Assays:

  • Proton translocation assays in reconstituted systems (liposomes with pH-sensitive dyes)

  • ATP synthesis coupling when combined with other subunits

  • Inhibitor binding studies (e.g., DCCD binding to functional Asp/Glu residue)

• Biophysical Properties:

  • Thermal stability profiling (functional protein shows cooperative unfolding)

  • Detergent/lipid binding characteristics (properly folded protein incorporates defined detergent/lipid molecules)

  • Protease resistance patterns (functional protein shows limited accessibility to specific sites)

When evaluating recombinant P. fluorescens atpE, apply these methods systematically and compare results with well-characterized reference samples whenever possible. This multi-parameter approach provides a comprehensive assessment of protein quality and functionality.

What are the best approaches for resolving data inconsistencies in P. fluorescens atpE research?

Resolving data inconsistencies in P. fluorescens atpE research requires systematic methodological approaches and careful experimental design:

• Source of Inconsistency: Protein Preparation Variability

  • Standardize expression conditions with detailed protocols

  • Implement batch validation using SDS-PAGE, Western blot, and activity assays

  • Prepare large, homogeneous protein batches for extended study series

  • Consider chemical biotinylation or other tagging methods for tracking specific preparations

• Source of Inconsistency: Assay Methodology Differences

  • Develop standardized assay protocols with detailed buffer compositions

  • Include internal controls and standards in each experiment

  • Perform calibration curves for quantitative measurements

  • Document equipment settings and environmental conditions

• Source of Inconsistency: Data Analysis Approaches

  • Establish consistent analysis parameters and software versions

  • Use blind analysis when applicable to reduce bias

  • Implement statistical approaches appropriate for the data type

  • Consider developing shared analysis pipelines within research communities

• Reconciliation Strategies:

  • Conduct head-to-head comparisons of different methods

  • Systematically vary one parameter at a time to identify critical variables

  • Employ orthogonal techniques to verify key findings

  • Collaborate with other laboratories to validate findings through inter-lab studies

When inconsistencies persist despite these approaches, consider alternative hypotheses such as different functional states of the protein, post-translational modifications, or interactions with specific lipids or other molecules that may be present in variable amounts across experiments. Documentation of all experimental conditions is crucial for resolving these types of inconsistencies, as seemingly minor variations in protocol may have significant impacts on membrane protein behavior.

How might structural studies of P. fluorescens atpE inform the development of novel antimicrobials?

Structural studies of P. fluorescens atpE offer promising avenues for antimicrobial development through several key research directions:

• Identification of Species-Specific Binding Sites:

  • High-resolution structural analysis can reveal unique pockets or interfaces in P. fluorescens atpE

  • Comparative analysis with human ATP synthase can identify bacterial-specific regions

  • Molecular docking studies can predict selective binding compounds

• Targeting c-ring Assembly and Stability:

  • Structural characterization of subunit interfaces may reveal targets to disrupt c-ring formation

  • Compounds that interfere with c-ring/a-subunit interaction could selectively inhibit bacterial ATP synthase

  • Small molecules that alter c-ring stability present novel antimicrobial mechanisms

• Proton Pathway Disruption:

  • Detailed mapping of the proton translocation pathway can identify critical residues

  • Structure-based design of molecules that block proton movement without affecting human ATP synthase

  • Allosteric modulators that alter proton binding/release kinetics

• Rational Design Based on Natural Inhibitors:

  • Structural studies of atpE bound to known inhibitors (oligomycin, DCCD) provide templates

  • Structure-activity relationship studies can guide optimization of lead compounds

  • Fragment-based approaches using structural data can identify novel chemotypes

Future structural studies should employ integrated approaches combining X-ray crystallography, cryo-EM, and NMR with computational methods to fully characterize P. fluorescens atpE in different functional states and in complex with potential inhibitors.

What novel applications of recombinant P. fluorescens atpE are emerging in biotechnology?

Recombinant P. fluorescens atpE is finding novel applications in biotechnology that leverage its unique structural and functional properties:

• Biosensor Development:

  • Integration of atpE into electrode systems for proton gradient detection

  • Fluorescently labeled atpE variants for monitoring membrane potential in real-time

  • Engineered atpE-based systems for detecting membrane-active compounds

• Bionanotechnology Platforms:

  • Self-assembling c-rings as templates for nanoparticle organization

  • atpE-based rotary nanomotors powered by proton gradients

  • Engineered protein pores based on c-ring architecture for controlled ion transport

• Drug Delivery Systems:

  • Reconstituted atpE in liposomes as pH-responsive drug release vehicles

  • Targeted delivery systems utilizing the proton-pumping capability

  • Fusion of therapeutic peptides to atpE for membrane targeting

• Bioenergetic Engineering:

  • Optimized atpE variants with enhanced ATP synthesis efficiency

  • Integration into artificial photosynthetic systems

  • Creation of hybrid energy-generating systems combining properties from different species

These emerging applications require interdisciplinary approaches combining protein engineering, nanotechnology, and synthetic biology. The development of optimized expression and purification protocols for functional atpE variants, as described in previous sections, provides the foundation for these biotechnological applications.

How does ATP synthase research in Pseudomonas species contribute to our understanding of bacterial evolution and adaptation?

Research on ATP synthase in Pseudomonas species provides valuable insights into bacterial evolution and adaptation through several key observations:

• Energetic Efficiency Adaptations:

  • Variations in c-ring stoichiometry across Pseudomonas species reflect adaptations to different energy environments

  • Changes in proton-binding site residues suggest adaptations to different pH habitats

  • Membrane composition interactions point to environmental specialization

• Regulatory Network Evolution:

  • Differences in ATP synthase gene organization between Pseudomonas species and other bacteria

  • Species-specific regulatory elements controlling ATP synthase expression

  • Co-evolution with other metabolic systems (similar to RNA degradosome evolution)

• Horizontal Gene Transfer and Recombination:

  • Evidence of horizontal gene transfer events in ATP synthase genes

  • Mosaic structures suggesting recombination between species

  • Conservation patterns revealing evolutionary constraints on specific domains

• Environmental Adaptation Signatures:

  • Cold-adapted Pseudomonas species show specific ATP synthase modifications

  • Clinical isolates versus environmental strains display different evolutionary patterns

  • Adaptations to oxygen limitation in biofilm-forming species

This evolutionary perspective provides context for interpreting structural and functional studies of P. fluorescens atpE. Understanding these adaptations can inform both fundamental microbiology and applications in synthetic biology where ATP synthase components might be engineered for specific functions or environments.