Recombinant Polaromonas sp. ATP synthase subunit c (atpE)

What is the ATP synthase subunit c (atpE) from Polaromonas sp.?

ATP synthase subunit c (atpE) from Polaromonas sp. is a critical component of the F0 sector of ATP synthase, forming the c-ring structure embedded in the membrane. This protein consists of 82 amino acids and functions as part of the rotary mechanism that couples proton translocation to ATP synthesis. The protein has several synonyms including F-type ATPase subunit c, F-ATPase subunit c, and lipid-binding protein, with the UniProt ID Q12GQ5 . The subunit is highly hydrophobic due to its membrane-spanning regions, which is evident from its amino acid sequence: MEHVLGFVALAAGLIIGLGAVGACIGIGIMGSKYLEAAARQPELMNELQTKMFLLAGLIDAAFLIGVGIAMMFAFANPFVLK .

How does the c-subunit contribute to ATP synthase function?

The c-subunit forms a ring structure (c₍ₙ₎ ring) in the membrane domain of ATP synthase, with the stoichiometry (n) varying between different organisms. The rotation of this c-ring is mechanically coupled to ATP synthesis through the γ-subunit, which extends into the F₁ catalytic region. As protons move through the membrane along an electrochemical gradient, they drive the rotation of the c-ring. This rotation is transmitted to the γ-subunit, causing conformational changes in the α₃β₃ head, where ATP synthesis occurs at the catalytic sites. For each complete rotation of the c-ring, 3 ATP molecules are synthesized, with the coupling ratio (protons translocated:ATP synthesized) determined by the number of c-subunits in the ring .

Why use recombinant expression for studying ATP synthase subunit c?

Recombinant expression of ATP synthase subunit c offers several advantages for research:

• Ability to obtain significant quantities of purified protein

• Introduction of specific mutations for structure-function studies

• Addition of tags (e.g., His-tag) to facilitate purification

• Control over expression conditions to optimize yield

• Opportunity to investigate the factors affecting c-ring stoichiometry and assembly

The recombinant approach also allows molecular biology techniques that cannot be applied to native c-rings, enabling deeper investigation into the structural and functional aspects of ATP synthase .

What expression systems are recommended for recombinant Polaromonas sp. atpE production?

For recombinant production of Polaromonas sp. atpE, E. coli expression systems have been successfully employed as demonstrated in the available commercial product . This is consistent with methodologies used for other ATP synthase c-subunits, such as those from spinach chloroplasts . The specific considerations for expression include:

For optimal results, researchers should consider expressing the protein with an N-terminal His-tag, which facilitates purification while maintaining protein functionality. Expression should be induced at mid-log phase (OD₆₀₀ ~0.6-0.8) with IPTG concentrations around 0.5-1.0 mM, followed by expression at reduced temperatures (16-25°C) to enhance proper folding of this membrane protein .

What purification strategies work best for recombinant atpE protein?

Purification of recombinant Polaromonas sp. atpE requires specialized techniques due to its hydrophobic nature as a membrane protein. A recommended multi-step approach includes:

• Cell lysis using sonication or high-pressure homogenization in buffer containing 20 mM Tris-HCl (pH 8.0) with protease inhibitors

• Membrane isolation through differential centrifugation

• Membrane protein extraction using detergents (e.g., DDM, LDAO, or Triton X-100)

• Immobilized metal affinity chromatography (IMAC) for His-tagged protein

• Size exclusion chromatography for final polishing

For quality control, SDS-PAGE analysis should confirm >90% purity, and circular dichroism analysis can verify the correct alpha-helical secondary structure expected for this protein . The purified protein is typically stored as a lyophilized powder or in solution with added glycerol (50%) at -20°C or -80°C to maintain stability, with aliquoting recommended to avoid repeated freeze-thaw cycles .

How can researchers investigate c-ring stoichiometry in ATP synthase?

Investigating c-ring stoichiometry is essential for understanding the proton-to-ATP coupling ratio and energetic efficiency of ATP synthase. Researchers can employ several complementary approaches:

• Recombinant Reconstitution Studies: Using purified recombinant c-subunits to reconstitute c-rings in vitro, followed by analysis of stoichiometry through analytical ultracentrifugation or native mass spectrometry

• Cryo-EM Analysis: High-resolution structural determination of assembled c-rings, allowing direct counting of subunits

• Cross-linking Studies: Chemical cross-linking of adjacent c-subunits, followed by mass spectrometric analysis to determine ring size

• Genetic Modification: Introducing mutations that affect c-c interactions to investigate factors influencing ring assembly

The stoichiometry of c-rings is known to vary across species (from c₁₀ to c₁₅), with corresponding changes in the coupling ratio (ions transported:ATP generated) from 3.3 to 5.0 . These variations may represent evolutionary adaptations to different energetic requirements or environmental conditions, making comparative studies particularly valuable.

What are the structural determinants of c-subunit assembly into functional rings?

The assembly of c-subunits into functional rings involves specific structural determinants that researchers should consider:

• Transmembrane Helices: The two alpha-helical domains in each c-subunit form the core structure that determines packing within the ring

• Conserved Residues: The essential proton-binding site typically involves a conserved acidic residue (Asp or Glu) in the C-terminal helix

• Inter-subunit Interactions: Hydrophobic interactions between adjacent subunits stabilize the ring structure

• Lipid Interactions: Specific lipid-protein interactions may influence assembly and stability of the c-ring

Research approaches to investigate these factors include site-directed mutagenesis of key residues, molecular dynamics simulations of c-ring assembly, and reconstitution studies in different lipid environments . Understanding these determinants could provide insights into the evolutionary diversity of c-ring stoichiometries and potential applications in synthetic biology.

How can proton translocation activity of reconstituted atpE be measured?

Measuring proton translocation activity of reconstituted Polaromonas sp. atpE requires specialized techniques to assess functionality in membrane environments:

• Proteoliposome Reconstitution: Incorporate purified recombinant atpE into liposomes with defined lipid composition:

  • Mix purified protein with lipids (typically phosphatidylcholine/phosphatidylethanolamine mixtures)

  • Remove detergent via dialysis or Bio-Beads

  • Confirm incorporation by density gradient centrifugation

• pH Sensitive Fluorescence Assays:

  • Load proteoliposomes with pH-sensitive fluorophores (ACMA, pyranine)

  • Create a pH gradient across the membrane

  • Monitor fluorescence changes upon addition of ionophores or ATP

• Patch-Clamp Electrophysiology:

  • For direct measurement of proton currents through reconstituted c-rings

  • Requires specialized equipment and expertise

• ATP Synthesis Coupling Assays:

  • Co-reconstitute c-subunits with complete F₁F₀ ATP synthase components

  • Measure ATP synthesis rates in response to artificially imposed proton gradients

These functional assays are critical for confirming that the recombinant protein maintains native-like activity and for investigating structure-function relationships .

What approaches can be used to study interactions between atpE and other ATP synthase components?

Understanding the interactions between atpE and other ATP synthase components is crucial for elucidating the assembly and function of the complete enzyme complex. Several techniques can be employed:

• Co-immunoprecipitation Studies:

  • Using antibodies against the His-tag or atpE itself

  • Identify interacting partners through mass spectrometry

• Surface Plasmon Resonance (SPR):

  • Immobilize purified atpE on sensor chips

  • Measure binding kinetics with other purified ATP synthase subunits

• Chemical Cross-linking Coupled with Mass Spectrometry:

  • Identify specific interaction sites between atpE and partner subunits

  • Particularly useful for examining interactions with a-subunit and peripheral stalk

• Förster Resonance Energy Transfer (FRET):

  • Label atpE and potential interacting partners with fluorophore pairs

  • Monitor distance-dependent energy transfer as evidence of interaction

• Electron Microscopy of Partially Assembled Complexes:

  • Visualize the spatial arrangement of atpE relative to other components

  • Can be combined with gold-labeling of specific subunits

These approaches provide complementary information about the structural organization and dynamic interactions within the ATP synthase complex .

How might Polaromonas sp. atpE contribute to understanding extremophile energy metabolism?

Polaromonas species are often found in extreme environments, including cold habitats such as glaciers and polar regions. The ATP synthase from these organisms likely has adaptations for function at low temperatures. Research directions include:

• Comparative Analysis: Structural comparison of Polaromonas sp. atpE with homologs from mesophilic organisms to identify cold-adaptation features

• Temperature-Dependent Activity Studies: Measuring ATP synthase activity across temperature ranges to determine thermal optima and stability

• Molecular Dynamics Simulations: Computational analysis of protein flexibility and conformational changes at different temperatures

• Lipid Composition Effects: Investigation of how membrane lipid composition affects c-ring assembly and function at low temperatures

• Evolutionary Analysis: Examination of sequence conservation patterns among psychrophilic and mesophilic ATP synthases

Understanding these adaptations could provide insights into bioenergetic flexibility in extreme environments and potentially inform biotechnological applications requiring low-temperature functionality .

Can recombinant atpE be used to investigate potential antibiotic targets in related pathogens?

ATP synthase has emerged as a potential antibiotic target in various bacterial pathogens. Recombinant Polaromonas sp. atpE could serve as a model system for investigating:

• Inhibitor Screening: Development of in vitro assays using reconstituted atpE to screen for specific inhibitors

• Structure-Based Drug Design: Using structural information to design molecules that specifically target bacterial ATP synthase subunit c

• Resistance Mechanism Studies: Investigation of how mutations in atpE might confer resistance to inhibitors

• Cross-Resistance Patterns: Examination of potential links between ATP synthase inhibition and metal/drug cross-resistance, as suggested by studies on P-type ATPases

The research could be particularly relevant given that some P-type ATPases (which share functional similarities with F-type ATP synthases) have been implicated in drug efflux mechanisms in bacteria like Mycobacterium tuberculosis . This connection between energy metabolism and antimicrobial resistance represents an important frontier in infectious disease research.

What strategies address the challenges of working with highly hydrophobic membrane proteins like atpE?

Working with hydrophobic membrane proteins like atpE presents specific technical challenges. Researchers can implement these strategies:

Combining these approaches can significantly improve success rates when working with challenging membrane proteins like atpE .

How can researchers troubleshoot issues with recombinant atpE protein folding and stability?

Proper folding and stability are critical challenges when working with recombinant membrane proteins. A systematic troubleshooting approach includes:

• Expression Condition Optimization:

  • Lower induction temperatures (16-20°C)

  • Reduced inducer concentrations

  • Extended expression times (overnight)

  • Addition of chemical chaperones to growth media

• Protein Quality Assessment:

  • Circular dichroism to confirm alpha-helical secondary structure

  • Thermal stability assays (DSF) to identify stabilizing conditions

  • Size exclusion chromatography to detect aggregation

  • Mass spectrometry to confirm intact protein

• Stability Enhancement:

  • Buffer optimization (pH, salt concentration, additives)

  • Addition of specific lipids during purification

  • Testing different detergent types and concentrations

  • Storage optimization (glycerol concentration, lyophilization conditions)

• Refolding Approaches:

  • Denaturation followed by controlled refolding in appropriate detergent/lipid mixtures

  • Step-wise dialysis to gradually remove denaturants

Regular monitoring of protein stability using analytical techniques throughout the purification process helps identify critical points where stability might be compromised .

What are the best structural characterization methods for recombinant atpE?

Structural characterization of membrane proteins like atpE requires specialized techniques. The most effective approaches include:

These methods provide complementary information and are often used in combination for comprehensive structural characterization .

How can researchers use molecular dynamics simulations to study atpE structure and function?

Molecular dynamics (MD) simulations offer powerful approaches to study membrane proteins like atpE at atomic resolution over time. Key applications include:

• Simulation Setup for Membrane Proteins:

  • Embedding the protein in a lipid bilayer matching experimental conditions

  • Addition of explicit water molecules and ions

  • Energy minimization and equilibration before production runs

• Specific Research Applications:

  • Proton translocation pathway identification

  • Conformational changes during rotation

  • Lipid-protein interactions at the c-ring periphery

  • Effects of mutations on structure and dynamics

  • Interactions between adjacent c-subunits within the ring

• Advanced Simulation Techniques:

  • Umbrella sampling for free energy calculations

  • Steered MD to study rotation mechanics

  • Coarse-grained simulations for longer timescales

  • QM/MM approaches for studying proton transfer reactions

• Integration with Experimental Data:

  • Validation of simulation results against experimental observables

  • Generation of testable hypotheses for experimental investigation

  • Interpretation of experimental results in atomic detail

MD simulations can provide insights into dynamic processes that are difficult to capture experimentally, complementing structural and functional studies of atpE .

How might synthetic biology approaches utilize recombinant atpE to create custom ATP synthases?

Synthetic biology offers exciting possibilities for engineering ATP synthases with novel properties using recombinant atpE as a building block:

• Altering c-Ring Stoichiometry:

  • Engineering c-subunits with modified interfaces to control ring size

  • Creating ATP synthases with predetermined coupling ratios

  • Optimizing energy conversion efficiency for specific applications

• Domain Swapping Approaches:

  • Creating chimeric proteins by combining domains from different species

  • Introducing properties from extremophiles (temperature resistance, pH tolerance)

  • Developing ATP synthases with novel regulatory properties

• Incorporation of Non-natural Amino Acids:

  • Introducing spectroscopic probes at specific positions

  • Creating photo-activatable variants for controlled function

  • Enhancing stability through novel chemical interactions

• Applications in Bionanotechnology:

  • Using c-rings as nanoscale rotary motors in synthetic devices

  • Creating artificial cells with customized bioenergetic properties

  • Developing sensors based on c-ring conformational changes

These approaches could lead to the development of ATP synthases with tailored properties for biotechnological applications, bioenergy research, and fundamental studies of energy transduction mechanisms .

What is the potential for using recombinant atpE in comparative studies of evolutionary adaptations in ATP synthases?

Recombinant atpE from Polaromonas sp. provides an excellent platform for evolutionary studies:

• Comparative Analysis Approaches:

  • Producing recombinant c-subunits from diverse species (extremophiles, mesophiles, thermophiles)

  • Systematically comparing structural features, stability, and functional properties

  • Correlating sequence variations with environmental adaptations

• Key Research Questions:

  • How does c-ring stoichiometry relate to environmental conditions?

  • What structural features determine thermal stability across diverse habitats?

  • How do ATP synthases balance efficiency vs. regulatory control in different organisms?

• Experimental Designs:

  • Ancestral sequence reconstruction and expression of predicted evolutionary precursors

  • Creation of chimeric proteins combining features from different evolutionary lineages

  • Functional characterization under varying conditions (temperature, pH, pressure)

• Integration with Genomic Data:

  • Correlation of ATP synthase variations with whole-genome adaptations

  • Analysis of co-evolution patterns between ATP synthase subunits

  • Investigation of horizontal gene transfer events affecting ATP synthase evolution

This research direction could provide fundamental insights into how this essential enzyme has evolved to support life across diverse environments, from psychrophilic to thermophilic conditions .