Recombinant Vibrio fischeri ATP synthase subunit c (atpE)

What is the structure and function of Vibrio fischeri ATP synthase subunit c (atpE)?

The ATP synthase subunit c in Vibrio fischeri is an 85-amino acid protein that forms part of the F0 sector of ATP synthase. The amino acid sequence (METLLSFSAIAVGIIVGLASLGTAIGFALLGGKFLEGAARQPEMAPMLQVKMFIIAGLLDAVPMIGIVIALLFTFANPFVGQLAG) reveals its highly hydrophobic nature, consistent with its role as a transmembrane component .

Functionally, the c-subunit forms an oligomeric ring (c-ring) that rotates relative to the a-subunit during ATP synthesis. This rotation is driven by proton translocation across the membrane, coupling the proton motive force to ATP synthesis. The glutamic acid residue at position 56 (E56) is particularly critical for proton binding and release during the rotational catalysis process .

Methodologically, researchers can study the structure using techniques such as X-ray crystallography, cryo-electron microscopy, or NMR spectroscopy of purified protein. Functional studies typically employ site-directed mutagenesis followed by biochemical assays measuring ATP synthesis or hydrolysis activities.

How is recombinant Vibrio fischeri ATP synthase subunit c (atpE) typically expressed in laboratory settings?

Recombinant V. fischeri ATP synthase subunit c is commonly expressed using E. coli expression systems. The gene encoding atpE can be cloned into expression vectors with appropriate affinity tags (such as an N-terminal 10×His tag) to facilitate purification .

For optimal expression, researchers should consider:

• Codon optimization for the expression host

• Use of specialized E. coli strains designed for membrane protein expression

• Induction conditions (temperature, inducer concentration, duration)

• Extraction and solubilization methods using detergents appropriate for membrane proteins

The expressed protein is typically provided in either liquid form in Tris/PBS-based buffer with 6% trehalose (pH 8.0) or as a lyophilized powder, both formats requiring storage at -20°C or -80°C to maintain stability .

What experimental approaches can verify proper folding and functionality of recombinant ATP synthase subunit c?

Verifying proper folding and functionality of recombinant V. fischeri ATP synthase subunit c requires multiple complementary approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to evaluate secondary structure

  • Size-exclusion chromatography to confirm oligomeric state

  • Limited proteolysis to assess tertiary structure

• Functional assays:

  • Reconstitution into liposomes and measurement of proton translocation

  • Assembly with other ATP synthase subunits to form functional F0 complexes

  • ATP synthesis/hydrolysis coupling measurements using pH-sensitive dyes or luminescence-based ATP detection

• Binding studies:

  • Isothermal titration calorimetry to measure binding of specific inhibitors

  • Fluorescence-based assays to detect conformational changes upon proton binding

These approaches provide complementary information about both structural integrity and functional capacity of the recombinant protein .

How do mutations in the c-subunit affect rotation and function of the F0F1-ATP synthase complex?

• Single mutations (e.g., cE56D in one subunit) reduce ATP synthesis activity but do not completely inhibit it

• Double mutations further decrease activity

• The spatial arrangement of mutations is critical - activity decreases as the distance between mutation sites increases

These findings reveal cooperative interactions among c-subunits during rotation. When analyzing the effects of mutations, researchers should consider:

• The specific residues mutated and their conservation across species

• The number and spatial distribution of mutations within the c-ring

• Effects on proton binding affinity, proton uptake kinetics, and conformational changes

Molecular dynamics simulations complement biochemical assays by revealing that:

• Prolonged proton uptake times in mutated c-subunits can be shared between subunits

• The degree of time-sharing decreases as the distance between mutation sites increases

• At least three c-subunits on the a/c interface cooperate during c-ring rotation

What methodologies are most effective for studying cooperation among c-subunits in ATP synthase?

Studying cooperation among c-subunits requires specialized approaches that can detect interactions between individual subunits within the c-ring. Effective methodologies include:

• Genetic fusion of c-subunits:
  Creating a genetically fused single-chain c-ring (as demonstrated with Bacillus PS3 ATP synthase) allows precise control over the number and position of mutations, enabling systematic investigation of cooperative effects .

• Site-specific mutagenesis:
  Introduction of mutations at specific positions (e.g., E56D) in different combinations and patterns within the c-ring to assess their impact on activity.

• Biochemical assays with mutation combinations:
  Measuring ATP synthesis, ATP hydrolysis, and proton pumping activities of enzymes with various mutation patterns to quantify cooperative effects.

• Proton transfer-coupled molecular dynamics simulations:
  Computational approaches that can model proton movement through the c-ring and predict how mutations affect proton uptake and release kinetics .

Analysis of such data should focus on:

• Activity patterns as a function of mutation position

• Cooperative effects that cannot be explained by simple additive models

• Correlation between simulation predictions and experimental observations

For example, studies with Bacillus PS3 ATP synthase revealed that ATP synthesis activity decreased further as the distance between two E56D mutations increased, providing clear evidence of functional coupling between neighboring c-subunits .

How does the membrane environment affect activity and stability of ATP synthase subunit c?

The membrane environment significantly influences ATP synthase subunit c function through:

• Lipid-protein interactions:

  • Specific lipids stabilize the c-ring structure and affect its rotational dynamics

  • The hydrophobic matching between membrane thickness and the hydrophobic region of subunit c impacts protein stability and function

  • Lipid head groups may influence proton access channels to key residues

• Membrane potential effects:

  • The proton motive force driving ATP synthesis depends on membrane potential

  • Local electric fields affect protonation states of key residues like E56

  • Membrane potential fluctuations can influence c-ring rotation kinetics

Methodological approaches to study these interactions include:

• Reconstitution of purified c-subunits into liposomes with defined lipid compositions

• Fluorescence-based assays to measure protein-lipid interactions

• Solid-state NMR to analyze structural changes in different membrane environments

• Molecular dynamics simulations incorporating realistic membrane models

When studying membrane effects, researchers should consider:

• Species-specific adaptations (V. fischeri thrives in marine environments)

• Temperature effects on membrane fluidity and protein stability

• Potential interactions with other membrane proteins or components

What are the challenges in studying proton translocation through the c-ring in Vibrio fischeri ATP synthase?

Studying proton translocation through the V. fischeri ATP synthase c-ring presents several significant challenges:

• Temporal resolution:
  Proton movement occurs on microsecond to millisecond timescales, requiring specialized techniques for detection.

• Spatial resolution:
  Identifying the precise pathway of protons through the protein structure requires atomic-level visualization.

• Coupling mechanisms:
  Distinguishing how proton movement couples to rotational motion and eventual ATP synthesis.

• Environmental sensitivity:
  Proton translocation depends on membrane potential, pH gradients, and lipid environment.

Methodological approaches to address these challenges include:

• Time-resolved spectroscopy to capture protonation state changes

• pH-sensitive fluorescent probes positioned at strategic locations

• Site-directed mutagenesis of key residues (particularly E56) involved in proton binding

• Hybrid quantum mechanics/molecular mechanics simulations that can model proton transfer events

A comprehensive experimental design would incorporate:

• Comparison of wild-type and mutant forms (such as E56D variants)

• Assessment under different pH and ionic conditions

• Correlation of proton translocation rates with ATP synthesis activity

• Integration of structural and functional data to develop mechanistic models

How can Vibrio fischeri ATP synthase research inform our understanding of bacterial adaptation to marine environments?

V. fischeri, as a luminescent, halophilic, gram-negative marine organism and bacterial symbiont of luminescent fish and squid, presents a unique model for studying ATP synthase adaptation to marine environments . Research approaches should consider:

• Evolutionary adaptations:

  • Comparison of V. fischeri ATP synthase sequences with non-marine bacteria

  • Identification of adaptive changes in salt-exposed regions of the protein

  • Analysis of how symbiotic relationships influenced ATP synthase evolution

• Functional adaptations:

  • Assessment of ATP synthase activity under varying salt concentrations

  • Measurement of proton binding/release kinetics in high-salt environments

  • Evaluation of c-ring stability in conditions mimicking marine habitats

• Ecological context:

  • Investigation of ATP synthase function in the context of bioluminescence energetics

  • Study of host-microbe interactions that may affect ATP synthase expression

  • Comparison of ATP synthase properties across different marine Vibrio species

Key methodological considerations include:

• Recreation of appropriate marine-like conditions in experimental systems

• Integration of ecological data with molecular findings

• Cross-species comparisons to identify convergent adaptations

The specific ecological niche of V. fischeri, which brings it into direct and intimate contact with eukaryotic hosts in marine environments, makes its ATP synthase particularly interesting for studying adaptation at the molecular level .