Recombinant Rhodospirillum rubrum ATP synthase subunit c (atpE)

What is the genetic organization of atpE within the ATP synthase operon of Rhodospirillum rubrum?

The atpE gene is part of the atp operon in Rhodospirillum rubrum, which contains a cluster of genes encoding the subunits of F1-ATPase. Transcriptional studies using S1 nuclease mapping and primer extension analysis have identified specific regulatory elements in this operon. The transcription start site is located at a guanine residue 236 bases upstream of the initiation codon of the first gene (delta-subunit) in the cluster . The promoter region shows similarities to Escherichia coli promoters but differs from those in Rhodopseudomonas blastica.

Transcription of the atp gene cluster appears to be coordinated, with the five genes being co-transcribed from a single promoter. A transcription termination site has been identified at a region of dyad symmetry followed by a run of thymidylate residues, which is characteristic of rho-independent transcriptional termination signals in E. coli .

What purification and refolding strategies are effective for recombinant Rhodospirillum rubrum ATP synthase subunits?

Based on research with other subunits of R. rubrum ATP synthase, the following methodological approach has proven effective and may be adapted for atpE :

Purification and Refolding Protocol:

• Solubilization of inclusion bodies using 8M urea

• Gradual dilution to reduce protein concentration (optimal at 50 μg/mL)

• Addition of high concentrations of MgATP (50 mM) during refolding

• Size-exclusion HPLC to separate monomeric from aggregated forms

• Functional validation through reconstitution experiments

This approach resulted in significant reduction of protein aggregation and increased recovery of functional protein. When implemented with the alpha subunit, a 50-60% decrease in aggregated forms was observed with parallel appearance of the monomeric peak .

How can functional activity of recombinant atpE be assessed?

Functional assessment of ATP synthase subunits requires integration into appropriate experimental systems. For R. rubrum ATP synthase subunits, the following methods have been effective:

• Reconstitution into beta-less R. rubrum chromatophores to measure ATP synthesis and hydrolysis restoration

• Assessment of subunit assembly using size-exclusion chromatography to detect formation of complexes

• Nucleotide binding assays to evaluate Mg-dependent binding of ATP and ADP

• ATPase activity measurements of reconstituted complexes

• Proton translocation assays using pH-sensitive fluorescent dyes

Research with alpha and beta subunits demonstrated that incubation of both monomers (which individually had no ATPase activity) resulted in the appearance of activity and assembled α₁β₁-dimers, indicating formation of a functional interface .

What factors influence the refolding efficiency of recombinant Rhodospirillum rubrum ATP synthase subunits?

Refolding efficiency of recombinant ATP synthase subunits from R. rubrum is influenced by multiple parameters that researchers should carefully optimize:

Research has shown that the refolding efficiency increases with decreasing protein concentrations and requires high concentrations of MgATP. In studies with the alpha subunit, refolding saturated at approximately 60% when 50 μg protein/mL was refolded in the presence of 50 mM MgATP .

Interestingly, different subunits show varying refolding behaviors. The beta subunit, when refolded under identical conditions to the alpha subunit, appeared almost exclusively as a monomer, suggesting subunit-specific folding requirements .

How does the assembly of ATP synthase subunits differ between recombinant systems and native environments?

Assembly studies with recombinant R. rubrum ATP synthase subunits have revealed important differences compared to native complexes:

• Recombinant alpha and beta subunits form α₁β₁-dimers but fail to assemble into the expected α₃β₃-hexamers, despite showing ATPase activity .

• The ATPase activity of these α₁β₁-dimers is comparable to that observed in isolated native chloroplast CF₁-α₃β₃, suggesting that these dimers contain only the catalytic nucleotide-binding site at their alpha/beta interface .

• The inability to form α₃β₃-hexamers appears to reflect lower stability of the noncatalytic alpha/beta interface in the recombinant system .

This indicates that while functional units can be recreated with recombinant subunits, the complete native architecture may require additional factors or stabilizing interactions that are missing in simplified recombinant systems.

How can atpE be utilized as a molecular target for bacterial detection and characterization?

While the search results focus primarily on Mycobacterium tuberculosis rather than R. rubrum, they provide valuable methodological insights for using atpE as a molecular target that could be adapted for R. rubrum research:

Primer Design Considerations for atpE Targeting:

• Optimal primer length: 18-24 nucleotides (longer primers >24 bases showed higher detection rates)

• Terminal nucleotide composition: The 3' end of primers should ideally end with G or C nucleotides to promote binding to target sites

• Specificity parameters: Careful design to differentiate between closely related species

In studies with M. tuberculosis, atpE primers designed using Thermo Fisher Scientific® software demonstrated 100% detection against positive control bacterial DNA, with a sensitivity of 61.54% and specificity of 100% compared to reference primers when tested against clinical samples .

What experimental approaches can address the challenges in structural characterization of membrane-embedded atpE?

Structural characterization of membrane proteins like atpE presents unique challenges requiring specialized approaches:

• Detergent Screening and Optimization:

  • Systematic testing of detergents for solubilization while maintaining native structure

  • Bicelles or nanodiscs as alternative membrane mimetics

• Reconstitution into Liposomes:

  • Functional validation through proton translocation assays

  • Assessment of oligomerization state in membrane environment

• Cryo-Electron Microscopy:

  • Single-particle analysis of purified complexes

  • Tomography of membrane-reconstituted samples

• Cross-linking Mass Spectrometry:

  • Identification of inter-subunit interactions

  • Validation of structural models

• Molecular Dynamics Simulations:

  • Investigation of proton translocation mechanisms

  • Prediction of mutation effects on structure and function

How do mutations in atpE affect proton translocation and ATP synthesis coupling?

While specific data on R. rubrum atpE mutations is not provided in the search results, a methodological framework for investigating such mutations would include:

• Site-Directed Mutagenesis Approach:

  • Target conserved residues involved in the proton-binding site

  • Alter the number of c-subunits per ring to investigate stoichiometry effects

  • Modify interface residues between c-subunits to study oligomerization

• Functional Assessment Methods:

  • Proton pumping assays using pH-sensitive fluorescent dyes

  • ATP synthesis/hydrolysis measurements in reconstituted systems

  • Thermodynamic coupling efficiency calculations

• Structural Validation Techniques:

  • Circular dichroism to verify secondary structure preservation

  • Cross-linking studies to assess oligomerization

  • NMR of isotopically labeled proteins to identify conformational changes