Recombinant Jannaschia sp. ATP synthase subunit c (atpE)

What is Jannaschia sp. ATP synthase subunit c (atpE)?

Jannaschia sp. ATP synthase subunit c (atpE) is a critical component of the F0 sector of the F-type ATP synthase in this marine α-proteobacterium of the Rhodobacterales order. This small hydrophobic protein (78 amino acids) forms part of the membrane-embedded c-ring structure that facilitates proton translocation across the membrane, driving ATP synthesis through rotational catalysis . The protein is also known by several synonyms including F-type ATPase subunit c and lipid-binding protein . As part of the F0F1 ATP synthase complex, subunit c plays a central role in the enzyme's function by participating in the mechanical coupling between proton flow and ATP production.

How is recombinant Jannaschia sp. ATP synthase subunit c typically expressed and purified?

Recombinant expression of Jannaschia sp. ATP synthase subunit c typically employs Escherichia coli as the expression host. The protein can be successfully expressed using the following methodology:

• Vector construction: The atpE gene is cloned with an N-terminal His-tag for purification purposes .

• Expression system: Transformed E. coli cells are grown under controlled conditions to optimize protein expression.

• Purification protocol:

  • Initial purification via affinity chromatography using the His-tag

  • Protein is typically eluted in a Tris/PBS-based buffer (pH 8.0)

  • Final preparation contains 6% trehalose as a stabilizing agent

After purification, the protein is typically lyophilized for long-term storage. For experimental use, reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL is recommended, with addition of 5-50% glycerol for long-term storage at -20°C/-80°C .

What storage conditions are optimal for maintaining the stability of recombinant Jannaschia sp. ATP synthase subunit c?

To maintain optimal stability of recombinant Jannaschia sp. ATP synthase subunit c, researchers should follow these evidence-based storage protocols:

• Short-term storage: Working aliquots can be maintained at 4°C for up to one week .

• Long-term storage: Store the lyophilized powder at -20°C to -80°C.

• After reconstitution: Add glycerol to a final concentration of 5-50% (50% being standard) and store in aliquots to avoid repeated freeze-thaw cycles .

• Handling precautions: Briefly centrifuge vials before opening to bring contents to the bottom.

The purified protein typically demonstrates greater than 90% purity as determined by SDS-PAGE, and maintaining this purity requires minimizing repeated freeze-thaw cycles, which can lead to protein degradation .

How does Jannaschia sp. ATP synthase subunit c function within the context of the complete ATP synthase complex?

In Jannaschia sp., as in other α-proteobacteria, the ATP synthase subunit c forms a multimeric ring (cn) in the membrane-embedded F0 sector of the ATP synthase complex. This c-ring functions as a critical component in the rotary mechanism of ATP synthesis through the following process:

• Protons flow through the c-ring along an electrochemical gradient

• Each proton translocation contributes to the rotation of the c-ring

• The c-ring rotation is mechanically coupled to the rotation of the γ-stalk in the F1 region

• This rotation drives conformational changes in the catalytic α3β3 head, catalyzing ATP synthesis

The stoichiometry of the c-ring (number of c subunits) varies between species (from c10 to c15 in characterized organisms) and directly determines the proton-to-ATP ratio, with each complete rotation of the c-ring generating 3 ATP molecules . Although the c-ring stoichiometry for Jannaschia sp. has not been definitively determined, research on related α-proteobacteria suggests potential patterns within phylogenetic groups.

What methods can be employed to study the inhibitory interactions between ζ subunits and ATP synthase in Jannaschia sp.?

Research has shown that the ζ subunit of Jannaschia sp. (Js-ζ) can inhibit ATP synthase activity, providing an important experimental model for studying regulatory mechanisms. Researchers can investigate these interactions using these methodological approaches:

• Heterologous reconstitution assays:

  • Express and purify recombinant Js-ζ

  • Reconstitute the protein with isolated F1FO-ATPase from related bacteria

  • Measure inhibitory activity using coupled ATPase assays

• Inhibition kinetics determination:

  • Conduct dose-response experiments with increasing concentrations of Js-ζ

  • Calculate apparent IC50 values through non-linear fitting to a non-competitive inhibitor model

  • Compare values to homologous systems (e.g., the appIC50 of Js-ζ for RcF1FO-ATPase was determined to be 1.12 μM)

• Binding site characterization:

  • Investigate the INGECORE binding site or α-DPβ-DPγ interface through mutational analysis

  • Employ structural biology techniques to visualize inhibitor-enzyme interactions

These approaches have revealed that Js-ζ is a potent inhibitor with nanomolar to micromolar affinities for ATP synthases from free-living α-proteobacteria .

How can researchers investigate the evolutionary relationships of ATP synthase subunit c across α-proteobacteria including Jannaschia sp.?

Investigating the evolutionary relationships of ATP synthase subunits provides valuable insights into functional conservation and adaptation. For Jannaschia sp. and other α-proteobacteria, researchers can employ these approaches:

• Phylogenetic analysis:

  • Construct phylogenetic trees based on sequence alignments of ATP synthase subunits

  • Compare evolutionary patterns between different orders (e.g., Rhodobacterales vs. Rhodospirillales)

  • Correlate phylogenetic relationships with functional conservation

• Functional conservation assessment:

  • Perform cross-species reconstitution experiments

  • Compare inhibitory potencies across closely and distantly related species

  • Examine structure-function relationships through sequence analysis

Research has demonstrated that Jannaschia sp. is closely related to other free-living α-proteobacteria like Paracoccus denitrificans, with evidence suggesting strong conservation of functional properties despite sequence divergence . The ζ subunit from Jannaschia sp. has evolved to preserve its inhibitory function despite exposure to diverse environmental conditions.

What are the methodological approaches for reconstituting recombinant Jannaschia sp. ATP synthase subunit c into functional c-rings?

Reconstitution of recombinant ATP synthase subunit c into functional c-rings represents a significant technical challenge. Based on approaches used with related systems, researchers can employ the following methodological framework:

• Purification of monomeric subunit c:

  • Express the protein with appropriate tags for purification

  • Employ detergent solubilization techniques optimized for hydrophobic membrane proteins

  • Achieve high purity through multi-step chromatography

• Assembly conditions optimization:

  • Screen different lipid compositions to mimic native membrane environments

  • Test various detergents and detergent-to-protein ratios

  • Evaluate the effect of pH, temperature, and ionic strength on assembly

• Functionality assessment:

  • Measure proton translocation activity in reconstituted proteoliposomes

  • Assess structural integrity through electron microscopy or atomic force microscopy

  • Determine stoichiometry using mass spectrometry or other biophysical techniques

While these methods have been applied to chloroplast ATP synthase c subunits, they can be adapted for Jannaschia sp. with appropriate modifications to account for the specific properties of this bacterial protein.

How does the structure of the ATP synthase c-ring influence the bioenergetics of Jannaschia sp. compared to other bacteria?

The structure of the ATP synthase c-ring directly impacts cellular bioenergetics through its influence on the proton-to-ATP ratio. For Jannaschia sp. and related bacteria, this relationship can be explored through:

• Stoichiometry determination:

  • The number of c subunits per ring (n) determines the H+/ATP ratio

  • In characterized organisms, this ratio ranges from 3.3 to 5.0, corresponding to c-rings with 10-15 subunits

  • Each c-ring rotation produces 3 ATP molecules regardless of size

• Environmental adaptation analysis:

  • Marine bacteria like Jannaschia sp. may have evolved specific c-ring structures in response to their environment

  • The c-ring stoichiometry can influence energy efficiency under different growth conditions

• Computational modeling:

  • Predict energetic consequences of different c-ring sizes

  • Calculate theoretical ATP yields under various proton motive force conditions

These approaches can help researchers understand how Jannaschia sp. has adapted its ATP synthase structure to thrive in its specific ecological niche as a marine α-proteobacterium.

What analytical techniques can be applied to study the interaction between ATP synthase subunit c and other membrane components in Jannaschia sp.?

Understanding the interactions between ATP synthase subunit c and other membrane components requires sophisticated analytical techniques:

• Crosslinking studies:

  • Use chemical crosslinkers to capture transient protein-protein or protein-lipid interactions

  • Identify interaction partners through mass spectrometry analysis

  • Map interaction surfaces using site-directed mutagenesis

• Native membrane analysis:

  • Isolate native membranes containing ATP synthase complexes

  • Characterize lipid-protein interactions using lipidomics

  • Assess the impact of membrane composition on ATP synthase activity

• Advanced microscopy:

  • Employ cryo-electron microscopy to visualize the ATP synthase in native-like environments

  • Use super-resolution microscopy to examine the distribution of ATP synthase complexes

  • Apply atomic force microscopy to study mechanical properties of reconstituted systems

These approaches can provide valuable insights into how Jannaschia sp. ATP synthase functions within its native membrane environment, potentially revealing adaptations specific to its marine habitat.