Recombinant Shewanella denitrificans ATP synthase subunit c (atpE)

What is the basic structure of ATP synthase subunit c (atpE) in Shewanella species?

ATP synthase subunit c (atpE) from Shewanella species is a small, highly hydrophobic membrane protein that forms the c-ring in the F₀ sector of ATP synthase. In Shewanella oneidensis, the full-length protein consists of 83 amino acids with the sequence: METILGMTAIAVALLIGMGALGTAIGFGLLGGKFLEGAARQPEMAPMLQVKMFIVAGLLDAVTMIGVGIALFMLFTNPLGAML . The protein has a predominantly alpha-helical structure that spans the membrane, with the c-subunits assembling into a ring structure that plays a critical role in proton translocation across the membrane. The hydrophobic nature of this protein reflects its location within the lipid bilayer of cellular membranes, where it functions as part of the rotary mechanism of ATP synthesis.

How does the c-subunit contribute to ATP synthesis in Shewanella?

The c-subunit forms a crucial component of the ATP synthase complex by creating a ring structure in the membrane-embedded F₀ portion. This c-ring participates in the rotary mechanism that couples proton translocation to ATP synthesis. When protons flow through the membrane down their electrochemical gradient, they interact with key residues in the c-subunits, causing rotation of the c-ring . This mechanical rotation drives conformational changes in the catalytic F₁ portion of the complex, facilitating ATP synthesis from ADP and inorganic phosphate. In Shewanella species, this process is particularly interesting as these bacteria can adapt to various environmental conditions, including different temperatures, which affects the directionality and efficiency of ATP synthase operation .

How do the thermodynamic constraints of ATP synthesis differ between Shewanella species and other bacteria?

Thermodynamic analysis reveals that Shewanella species have evolved specific adaptations in their ATP synthase function compared to other bacteria. Studies of Shewanella species have shown temperature-dependent metabolic responses that affect ATP synthase operation. For instance, in cold-adapted Shewanella strains, the ATP synthase reaction (ATPS4r) exhibits different directionalities depending on temperature conditions. At lower temperatures (4°C), the ATP synthase may not carry significant flux in the ATP-producing direction, potentially reflecting an ATP conservation mechanism . This contrasts with operation at 15°C or 20°C, where the enzyme functions more conventionally.

These differences represent metabolic adaptations to environmental conditions, with psychrophilic Shewanella species showing higher [ATP]/[ADP] ratios at lower temperatures despite reduced growth rates. These thermodynamic adaptations likely contribute to the ecological success of Shewanella in diverse environments, particularly cold marine habitats .

What computational approaches are most effective for modeling Shewanella atpE structure and function?

Effective computational modeling of Shewanella atpE involves a multi-step approach similar to that used for other bacterial ATP synthase components:

• Homology modeling: Using known structures (such as ATP synthases from E. coli or other bacteria) as templates for constructing the three-dimensional structure based on sequence alignment. This approach has been successfully applied to AtpE from other species with tools like Modeller9.16 .

• Energy minimization and refinement: The initial model requires energy minimization to remove steric clashes and optimize geometry. Molecular dynamics simulations (10+ ns) using tools like AMBERTOOLS10 can refine the structure to a stable conformation .

• Model validation: Assessment using Ramachandran plots, ERRAT, and Verify_3D to evaluate stereochemical quality and structural reliability. A successful model should have low RMSD values when superimposed with template structures (ideally <1Å) .

• Molecular docking studies: For studying interactions between atpE and potential ligands or inhibitors, tools like AutoDock4.2 can be employed with grid maps set at appropriate dimensions (e.g., 60 × 60 × 60 with 0.375 Å spacing) .

These approaches can reveal critical insights into the structure-function relationship of Shewanella atpE, particularly when combined with experimental validation.

How does the c-subunit in Shewanella ATP synthase contribute to the kinetic advantage of the rotary mechanism?

The c-subunit ring is central to the kinetic advantage of the rotary mechanism in ATP synthesis. Comparative biophysical analysis has demonstrated that the rotary mechanism outperforms alternative ATP synthesis mechanisms, particularly under low-energy conditions . In Shewanella species, the c-ring structure facilitates sequential proton binding and transport across the membrane, allowing for more efficient energy coupling than simultaneous multi-proton transport mechanisms.

The exact arrangement of c-subunits in the ring creates a pathway for proton translocation that optimizes the use of the proton motive force. Mathematical modeling using nonequilibrium steady-state (NESS) analysis has shown that this sequential mechanism provides superior performance over a broad range of physiological and pathological conditions . The architecture of the c-ring, including the specific arrangement of proton-binding sites, contributes significantly to this kinetic advantage by allowing for efficient energy conversion even when the driving force (proton gradient) is minimal.

What are the structural and functional differences between atpE from Shewanella and other bacterial species?

While the core structure and function of ATP synthase subunit c are conserved across bacterial species, Shewanella atpE exhibits specific adaptations that reflect its ecological niche. Comparative analysis reveals several key differences:

These differences reflect evolutionary adaptations to different environmental conditions. For instance, cold-adapted Shewanella species show modifications in their ATP synthase that allow for efficient operation at lower temperatures, including potential changes in the c-ring architecture and proton-binding properties. These adaptations may involve subtle changes in amino acid composition that affect protein flexibility, hydrophobicity, and interaction with the surrounding membrane environment .

What is the recommended protocol for expressing and purifying recombinant Shewanella atpE protein?

Based on established protocols for Shewanella proteins, the following methodology is recommended for expressing and purifying recombinant Shewanella atpE:

• Expression System Selection: E. coli is the preferred expression system due to its efficiency and compatibility. BL21(DE3) or similar strains are recommended for membrane protein expression .

• Construct Design:

  • Include an N-terminal His-tag for purification

  • Ensure the full coding sequence (amino acids 1-83) is included

  • Optimize codon usage for E. coli expression

  • Consider using a pET-based vector with T7 promoter

• Expression Conditions:

  • Culture at 30°C until OD600 reaches 0.6-0.8

  • Induce with 0.5mM IPTG

  • After induction, lower temperature to 18-20°C for overnight expression

  • Use rich media (such as Terrific Broth) supplemented with glucose

• Membrane Isolation:

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

  • Resuspend in buffer containing 50mM Tris-HCl pH 8.0, 200mM NaCl, 5mM MgCl₂

  • Disrupt cells by sonication or French press

  • Remove unbroken cells by centrifugation (10,000×g, 20 minutes, 4°C)

  • Isolate membranes by ultracentrifugation (150,000×g, 1 hour, 4°C)

• Protein Extraction and Purification:

  • Solubilize membranes in buffer with 1% n-dodecyl-β-D-maltopyranoside (DDM)

  • Clarify by centrifugation (150,000×g, 30 minutes, 4°C)

  • Purify using Ni-NTA affinity chromatography

  • Elute with imidazole gradient (50-300mM)

  • Further purify by size-exclusion chromatography in buffer containing 0.05% DDM

• Storage:

  • Store as lyophilized powder or in buffer with 50% glycerol at -80°C

  • Avoid repeated freeze-thaw cycles

This protocol can be modified based on specific experimental requirements and the intended use of the purified protein.

How can researchers effectively reconstitute purified atpE into liposomes for functional studies?

Reconstitution of atpE into liposomes requires careful handling due to its hydrophobic nature. The following methodology is recommended:

• Liposome Preparation:

  • Prepare lipid mixture (typically 3:1 phosphatidylcholine:phosphatidic acid)

  • Dissolve lipids in chloroform, dry under nitrogen, and then vacuum for 2-3 hours

  • Rehydrate with buffer (10mM HEPES pH 7.5, 100mM KCl) to 10mg/ml concentration

  • Subject to 5 freeze-thaw cycles

  • Extrude through 400nm polycarbonate filters

• Protein Incorporation:

  • Solubilize the purified atpE protein in 0.5% DDM or other suitable detergent

  • Mix with prepared liposomes at protein:lipid ratio of 1:100 (w/w)

  • Add Bio-Beads SM-2 (80mg/ml) to remove detergent

  • Incubate with gentle agitation for 2 hours at room temperature

  • Add fresh Bio-Beads and continue incubation overnight at 4°C

  • Remove Bio-Beads by gentle centrifugation

• Verification of Incorporation:

  • Analyze by freeze-fracture electron microscopy or dynamic light scattering

  • Check protein orientation by protease protection assay

  • Confirm functionality by proton pumping assays using pH-sensitive fluorophores

• Functional Assessment:

  • For proton translocation studies, prepare liposomes with pH-sensitive fluorophores (e.g., ACMA or pyranine)

  • For ATP synthesis studies, include ATP detection systems within or outside liposomes

  • Measure activity under various conditions to assess temperature dependence and other factors

This methodology allows for functional studies of the c-subunit in a controlled membrane environment, enabling detailed investigation of its role in proton translocation and ATP synthesis.

What techniques are most effective for studying the structure and interactions of the c-ring in Shewanella ATP synthase?

Multiple complementary techniques can be employed to study the structure and interactions of the c-ring:

• Cryo-Electron Microscopy (Cryo-EM):

  • Most powerful for resolving the complete ATP synthase structure in different rotational states

  • Can achieve near-atomic resolution to visualize c-ring assembly and interactions

  • Sample preparation involves purification of intact ATP synthase complex and vitrification

• X-ray Crystallography:

  • Provides high-resolution structural data when crystals can be obtained

  • May require specialized crystallization conditions for membrane proteins

  • Often used for isolated c-rings rather than complete ATP synthase complexes

• Cross-linking Studies:

  • Identify interaction partners and orientation within the complex

  • Use chemical cross-linkers followed by mass spectrometry

  • Can be performed in native membranes or reconstituted systems

• Molecular Dynamics Simulations:

  • Model conformational changes and proton pathways

  • Investigate interactions with lipids and other subunits

  • Typically requires 100+ ns simulations with specialized force fields for membrane proteins

• Site-Directed Spin Labeling (SDSL) with EPR:

  • Measures distances between specific residues

  • Provides information about conformational changes during function

  • Requires introduction of cysteine residues for spin label attachment

• Single-Molecule FRET:

  • Monitors rotational movements during operation

  • Requires strategic placement of fluorophores

  • Can capture dynamic processes in real-time

• Native Mass Spectrometry:

  • Determines stoichiometry and stability of the c-ring assembly

  • Requires specialized ionization techniques for membrane proteins

  • Can verify the number of c-subunits in the ring

Each of these techniques provides unique insights, and their combination offers a comprehensive understanding of c-ring structure and function in Shewanella ATP synthase.

What are common challenges in working with recombinant atpE and how can they be addressed?

Working with recombinant atpE presents several challenges due to its hydrophobic nature and membrane protein characteristics:

Additionally, freezing and thawing can disrupt the protein structure. It is recommended to store the protein with 6% trehalose in Tris/PBS-based buffer (pH 8.0) and reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for long-term storage at -20°C/-80°C .

How can researchers differentiate between functional and non-functional atpE in experimental settings?

Differentiating between functional and non-functional atpE requires multiple assessment approaches:

• Proton Translocation Assays:

  • Reconstitute purified atpE into liposomes containing pH-sensitive fluorophores

  • Monitor fluorescence changes upon establishment of proton gradients

  • Functional atpE will facilitate proton movement, causing detectable fluorescence changes

  • Compare results to known inhibitors of c-ring function (e.g., DCCD) as controls

• ATP Synthesis Activity Measurements:

  • Incorporate atpE into complete ATP synthase complexes

  • Apply artificial proton gradients and measure ATP production

  • Use luciferase-based assays for sensitive detection of ATP synthesis

  • Functional atpE will enable ATP production proportional to the proton gradient

• Structural Assessment:

  • Analyze oligomeric state by native PAGE or size-exclusion chromatography

  • Functional atpE forms proper c-rings while non-functional forms may appear as monomers or irregular aggregates

  • Examine secondary structure by circular dichroism spectroscopy

  • Functional protein should show characteristic alpha-helical patterns

• Binding Studies with Known Interactors:

  • Test interaction with a and b subunits of the ATP synthase

  • Perform co-immunoprecipitation or pull-down assays

  • Functional atpE should demonstrate specific binding to these partners

• Response to Inhibitors:

  • Treat with specific c-subunit inhibitors like dicyclohexylcarbodiimide (DCCD)

  • Monitor changes in any of the above assays

  • Functional atpE will show dose-dependent inhibition

These multiple approaches provide complementary information about atpE functionality and should be used in combination for comprehensive assessment.

What are the implications of c-subunit research for understanding bioenergetic adaptations in Shewanella species?

Research on ATP synthase c-subunit provides critical insights into how Shewanella species adapt their bioenergetic systems to diverse environmental conditions:

• Temperature Adaptation Mechanisms:
  Studies of Shewanella ATP synthases reveal temperature-dependent metabolic shifts that affect ATP production efficiency. At lower temperatures, some Shewanella species show increased [ATP]/[ADP] ratios despite reduced growth rates, indicating special adaptations in their ATP synthase operation that may involve specific c-ring configurations .

• Energy Conservation Strategies:
  The c-ring architecture in psychrophilic Shewanella appears optimized for energy conservation under challenging conditions. Models indicate that the ATP synthase reaction (ATPS4r) may operate differently at low temperatures (4°C) compared to moderate temperatures (15-20°C), potentially reflecting a mechanism to preserve cellular energy in cold environments .

• Proton-Motive Force Utilization:
  The efficiency with which Shewanella ATP synthase converts proton-motive force into ATP synthesis reveals adaptations to environments with varying energy availability. The rotary mechanism involving the c-ring provides kinetic advantages over alternative mechanisms, particularly under low-energy conditions often encountered in Shewanella habitats .

• Environmental Niche Specialization:
  The specific adaptations in c-subunit structure and function contribute to Shewanella's ability to colonize diverse environments, from deep-sea sediments to freshwater systems. These adaptations may include modifications in proton-binding residues, c-ring size, or interaction with other ATP synthase components .

• Electron Transport Chain Integration:
  The c-ring's role in coupling electron transport to ATP synthesis reflects Shewanella's remarkable respiratory versatility, including its ability to use diverse electron acceptors. Understanding this coupling mechanism provides insights into how these bacteria can thrive in anaerobic environments using various terminal electron acceptors .

These insights contribute to our understanding of microbial adaptation to extreme environments and offer potential applications in biotechnology, particularly for processes requiring energy efficiency under challenging conditions.

How might the study of Shewanella atpE contribute to the development of new antimicrobial strategies?

The study of Shewanella atpE has several implications for antimicrobial development:

• Target Identification and Validation:
  ATP synthase is an essential enzyme for bacterial survival, making it an attractive antimicrobial target. Research on Shewanella atpE structure and function contributes to understanding bacterial ATP synthases more broadly. The c-subunit, in particular, has been identified as a potential drug target in other bacterial species, with inhibitors like bedaquiline already developed for Mycobacterium tuberculosis AtpE .

• Structure-Based Drug Design:
  Detailed structural models of Shewanella atpE, developed through homology modeling and validated experimentally, provide templates for computational drug screening. Using approaches similar to those documented for Mycobacterium tuberculosis AtpE, researchers can identify compounds that bind to the enzyme with minimum binding energy . The most promising candidates would show strong binding to bacterial AtpE while having minimal interaction with human ATP synthase components.

• Comparative Analysis Across Species:
  Studying the c-subunit from Shewanella allows for identification of conserved features across bacterial ATP synthases that could serve as broad-spectrum targets, as well as species-specific features that might enable selective targeting. The identification of compounds with binding energies in the range of -8.69 to -8.44 kcal/mol (lower than ATP's binding energy) represents a promising approach for developing effective inhibitors .

• Novel Inhibition Mechanisms:
  Research into the functional mechanisms of ATP synthase c-subunit reveals potential inhibition strategies beyond direct binding site competition. For example, compounds that disrupt c-ring assembly or rotation, interfere with proton binding, or alter the interaction between the c-ring and other subunits could represent novel antimicrobial approaches.

These investigations contribute to addressing the growing challenge of antimicrobial resistance by identifying new targets and mechanisms for antimicrobial development.

What are the most promising directions for future research on Shewanella ATP synthase subunit c?

Several promising research directions emerge from current understanding of ATP synthase subunit c:

• Environmental Adaptation Mechanisms:
  Further investigation into how Shewanella species modify their ATP synthase function across environmental gradients (temperature, pH, salinity) could reveal novel adaptative mechanisms. Comparative studies of atpE across Shewanella species from different habitats would be particularly valuable in understanding these adaptations .

• Synthetic Biology Applications:
  The unique properties of Shewanella atpE, particularly its ability to function under diverse conditions, make it an interesting component for synthetic biology applications. Engineered ATP synthases incorporating Shewanella components could enable energy production in artificial systems under challenging conditions.

• Integration with Electron Transport Systems:
  Shewanella species are known for their diverse respiratory capabilities, including extracellular electron transfer. Research connecting ATP synthase function to these unique electron transport systems could reveal insights into energy conservation in these metabolically versatile bacteria.

• Single-Molecule Studies:
  Advanced biophysical techniques like single-molecule FRET or high-speed AFM could provide unprecedented insights into the dynamics of c-ring rotation and proton translocation in real-time, building on existing understanding of ATP synthase kinetics .

• Structural Comparison Across Evolutionary Timescales:
  Detailed structural analysis of atpE across diverse organisms could reveal evolutionary adaptations in this essential machinery. Shewanella, with its environmental adaptability, represents an interesting model for such comparative studies .

These research directions promise to advance both fundamental understanding of bioenergetics and applied aspects of bacterial physiology with potential biotechnological applications.