Recombinant Escherichia coli O8 ATP synthase subunit c (atpE)

What is the structural composition of recombinant E. coli O8 ATP synthase subunit c?

Recombinant E. coli O8 ATP synthase subunit c (atpE) is a relatively small protein consisting of 79 amino acids with the sequence: MENLNMDLLYMAAAVMMGLAAIGAAIGIGILGGKFLEGAARQPDLIPLLRTQFFIVMGLVDAIPMIAVGLGLYVMFAVA . The protein is highly hydrophobic, with multiple transmembrane segments that form a ring-like structure in the F0 sector of ATP synthase. When produced recombinantly, it is often expressed with an N-terminal His-tag to facilitate purification and experimental manipulation. The protein is typically purified in a detergent-solubilized form due to its hydrophobic nature, which requires careful consideration of buffer conditions to maintain stability while preserving native conformation.

How does ATP synthase subunit c contribute to the chemiosmotic mechanism of ATP synthesis?

ATP synthase subunit c plays a crucial role in the chemiosmotic mechanism by forming a ring in the membrane-embedded F0 sector that facilitates proton translocation. In experimental demonstrations of the chemiosmotic mechanism, researchers have shown that a proton gradient across a membrane can drive ATP synthesis even without light (in photosynthetic systems) . The c-ring rotates as protons pass through the F0 complex, with each c subunit binding and releasing protons as it rotates. This rotation is mechanically coupled to conformational changes in the F1 sector that catalyze ATP synthesis. Mutation studies targeting key residues in subunit c have demonstrated its essential role in proton conductance and the coupling of proton movement to rotational energy.

What experimental systems are typically used to study recombinant E. coli O8 ATP synthase subunit c?

Several experimental systems have been developed to study the recombinant E. coli O8 ATP synthase subunit c:

These systems allow researchers to investigate various aspects of subunit c function, from basic structural properties to complex mechanistic details of ATP synthesis .

How can researchers investigate subunit rotation in ATP synthase using recombinant E. coli O8 ATP synthase subunit c?

Investigating subunit rotation in ATP synthase requires sophisticated experimental approaches. One established method involves engineering the c subunit with specific tags or modifications. For example, researchers have introduced a His-tag at the N-terminus of the c subunit (after Met-1) to facilitate attachment of probes . The key methodological steps include:

• Engineering plasmids carrying modified ATP synthase genes, such as pBWU13 which contains all E. coli F0F1 genes

• Introducing specific modifications, such as replacing Glu-2 of the c subunit with His to create restriction sites

• Adding biotin-binding domains to specific subunits (β or a) for attachment to surfaces

• Using fluorescent or gold nanoparticle probes attached to the c-ring

• Employing single-molecule microscopy techniques to observe rotation directly

This approach has enabled researchers to observe the rotational motion of the c-ring relative to other subunits, providing critical insights into the mechanics of ATP synthesis .

What is the significance of atpE mutations in antibiotic resistance research, particularly for tuberculosis treatment?

The atpE gene has emerged as an important target in antibiotic resistance research, particularly in Mycobacterium tuberculosis studies. Researchers investigating bedaquiline (BDQ) resistance have identified specific mutations in the atpE gene that may affect drug susceptibility:

• The Ile66Val mutation (c.196A>G) in atpE has been reported in clinical isolates, though interestingly, this particular mutation did not confer significant resistance to BDQ in some studies .

• Comprehensive analysis methods include:

  • PCR amplification of the atpE gene from clinical isolates

  • Sanger sequencing to identify potential mutations

  • Comparison to wild-type M. tuberculosis H37Rv sequences

  • Site-directed mutagenesis to introduce specific mutations for functional validation

  • Minimum inhibitory concentration (MIC) assays to quantify resistance levels

These studies are critical for understanding mechanisms of drug resistance and developing strategies to overcome treatment failures in tuberculosis patients .

How do structural variations in ATP synthase subunit c affect function across different bacterial species?

Structural variations in ATP synthase subunit c across bacterial species can significantly impact function and have important evolutionary and therapeutic implications. While the E. coli O8 subunit c consists of 79 amino acids, homologs in other species vary in length and sequence composition. Research methodologies to investigate these variations include:

• Comparative genomic analyses to identify conserved and variable regions

• Homology modeling based on known structures

• Heterologous expression systems to produce and study subunit c from different species

• Functional complementation assays to test interchangeability between species

• Biochemical assays measuring ATP synthesis rates with hybrid complexes

What are the optimal protocols for expressing and purifying recombinant E. coli O8 ATP synthase subunit c?

Expression and purification of recombinant E. coli O8 ATP synthase subunit c requires careful optimization due to its hydrophobic nature. Based on established protocols, the following methodological approach is recommended:

Expression System:

• Use E. coli as the expression host (typically BL21(DE3) strains)

• Culture in rich medium such as LB or 2YT at 37°C

• Induce expression at OD600 of 0.6-0.8 with IPTG (0.5-1 mM)

• Allow expression for 3-4 hours or overnight at reduced temperature (25-30°C)

Purification Protocol:

• Harvest cells by centrifugation (5,000 × g, 10 minutes)

• Resuspend in lysis buffer containing:

  • 50 mM Tris-HCl, pH 8.0

  • 200 mM NaCl

  • 1% detergent (typically DDM or LDAO)

  • Protease inhibitor cocktail

• Lyse cells by sonication or French press

• Centrifuge (20,000 × g, 30 minutes) to remove cell debris

• Purify using Ni-NTA affinity chromatography (if His-tagged)

• Elute with imidazole gradient (50-300 mM)

• Further purify by size exclusion chromatography

• Store in buffer containing 0.05% detergent and 6% trehalose

For long-term storage, it is recommended to add 50% glycerol and store at -20°C/-80°C in aliquots to avoid repeated freeze-thaw cycles .

How can site-directed mutagenesis be designed to study specific residues in ATP synthase subunit c?

Site-directed mutagenesis is a powerful approach to investigate structure-function relationships in ATP synthase subunit c. Based on established research methodologies, the following approach is recommended:

Primer Design for Site-Directed Mutagenesis:

• Design complementary primers (25-45 nucleotides) that contain the desired mutation centrally positioned

• Ensure primers have a GC content of 40-60% and terminate in one or more C or G bases

• Aim for a melting temperature (Tm) of ≥78°C

Mutagenesis Protocol:

• Use a high-fidelity DNA polymerase (e.g., Pfu Ultra or Q5)

• Set up PCR with:

  • Template plasmid containing wild-type atpE gene

  • Mutagenic primers

  • dNTPs, buffer, and polymerase

• Perform PCR with an extended extension time

• Digest with DpnI to remove template DNA

• Transform into competent E. coli cells

• Select transformants and verify by sequencing

For more complex approaches such as homologous recombineering, specialized techniques may be employed:

• Introduce a plasmid expressing recombinase protein (e.g., gp61 encoded by Che9c)

• Induce recombinase expression (e.g., with acetamide)

• Transform with single-stranded DNA oligonucleotide carrying the mutation

• Select transformants using appropriate markers

• Verify mutations by PCR and sequencing

This approach has been successfully used to create point mutations such as c.196A>G (Ile66Val) in the atpE gene for functional studies .

What techniques can be used to measure the functionality of recombinant ATP synthase subunit c in vitro?

Several techniques can be employed to assess the functionality of recombinant ATP synthase subunit c in vitro:

For the rotational assay specifically, researchers have successfully employed a technique where:

• The c subunit is modified with a His-tag at the N-terminus

• Other subunits (β or a) are tagged with biotin-binding domains

• The complex is reconstituted into a membrane or attached to a surface

• Rotation is observed using fluorescence microscopy or other imaging techniques

These techniques collectively provide a comprehensive assessment of ATP synthase subunit c functionality.

How is recombinant E. coli O8 ATP synthase subunit c employed in drug discovery research?

Recombinant E. coli O8 ATP synthase subunit c serves as an important tool in drug discovery research, particularly for antibacterial agents. Methodological approaches include:

• Target-based screening:

  • Reconstituted subunit c or c-rings are used to screen compound libraries

  • Functional assays measuring ATP synthesis inhibition identify potential hits

  • Binding assays (e.g., surface plasmon resonance) quantify interaction strength

• Structure-based drug design:

  • X-ray crystallography or cryo-EM structures of the c-ring guide compound optimization

  • In silico docking studies identify potential binding sites

  • Rational design of analogs based on structure-activity relationships

• Resistance studies:

  • Site-directed mutagenesis of key residues to mimic potential resistance mutations

  • Selection of resistant mutants through serial passage in sub-inhibitory concentrations

  • Cross-resistance profiling against known ATP synthase inhibitors

This approach has been instrumental in understanding the molecular basis of action for drugs targeting ATP synthase, such as bedaquiline for tuberculosis treatment, and in identifying potential new therapeutic candidates.

What is the significance of atpE mutations in Mycobacterium tuberculosis, and how can recombinant protein studies inform tuberculosis treatment?

The atpE gene in Mycobacterium tuberculosis has gained significant research attention due to its role as the target of bedaquiline (BDQ), a novel antituberculosis drug. Studies examining atpE mutations provide critical insights for tuberculosis treatment:

• Mutation profiling methodologies:

  • PCR amplification and sequencing of the atpE gene from clinical isolates

  • Comparison to wild-type M. tuberculosis H37Rv sequences

  • Correlation of specific mutations with phenotypic resistance profiles

• Functional validation approaches:

  • Site-directed mutagenesis to introduce specific mutations (e.g., Ile66Val)

  • Expression of mutant proteins for biochemical characterization

  • Drug susceptibility testing to determine minimum inhibitory concentrations

• Structural and mechanistic studies:

  • Production of recombinant wild-type and mutant atpE proteins

  • Binding studies to quantify drug-protein interactions

  • Structural analyses to identify conformational changes induced by mutations

These studies have revealed that while some atpE mutations (like Ile66Val) have been reported in clinical isolates, they don't always confer significant resistance to BDQ, suggesting complex resistance mechanisms involving other genes such as Rv0678 . This research is essential for monitoring and addressing emerging resistance to bedaquiline, one of the few new antituberculosis drugs developed in recent decades.

How does research on E. coli ATP synthase subunit c inform studies on human mitochondrial ATP synthase disorders?

Research on E. coli ATP synthase subunit c provides valuable insights into human mitochondrial ATP synthase disorders through comparative studies. The methodological approaches include:

• Comparative structural analysis:

  • Alignment of bacterial and human ATP synthase subunit sequences

  • Identification of conserved residues and domains

  • Homology modeling to predict effects of human mutations

• Functional conservation studies:

  • Expression of human subunits in bacterial systems

  • Creation of hybrid ATP synthase complexes

  • Assessment of cross-species functional complementation

• Disease mutation modeling:

  • Introduction of human disease-associated mutations into bacterial homologs

  • Biochemical characterization of mutant proteins

  • Correlation of functional defects with clinical presentations

These approaches leverage the experimental tractability of bacterial systems to understand more complex eukaryotic ATP synthases. The high degree of conservation in the catalytic mechanism makes E. coli a valuable model organism for studying fundamental aspects of ATP synthase function that may be disrupted in human mitochondrial disorders.

What emerging technologies show promise for studying ATP synthase subunit c dynamics?

Several cutting-edge technologies are enhancing our ability to study ATP synthase subunit c dynamics with unprecedented resolution:

• Cryo-electron microscopy (cryo-EM):

  • Enables visualization of ATP synthase in different conformational states

  • Allows determination of c-ring stoichiometry across species

  • Provides insights into subunit interactions during rotation

• Advanced fluorescence techniques:

  • Single-molecule FRET to monitor conformational changes

  • Super-resolution microscopy to visualize subunit arrangement

  • Time-resolved fluorescence to capture transient states

• Computational approaches:

  • Molecular dynamics simulations of the complete ATP synthase complex

  • Quantum mechanical calculations of proton transfer events

  • Machine learning for prediction of mutation effects

These technologies are expected to resolve long-standing questions about the precise mechanism of proton translocation through the c-ring and how this energy is transduced into rotational motion and ultimately ATP synthesis.

How might synthetic biology approaches utilize recombinant ATP synthase subunit c to create novel energy-harvesting systems?

Synthetic biology offers exciting possibilities for engineering ATP synthase subunit c for biotechnological applications:

• Designer c-rings with altered properties:

  • Modified proton binding sites to operate at different pH ranges

  • Engineered c-rings with different stoichiometries for varied bioenergetic efficiencies

  • Introduction of non-canonical amino acids for novel functionality

• Integration into artificial systems:

  • Incorporation into synthetic vesicles for ATP production

  • Creation of hybrid organic-inorganic interfaces for energy harvesting

  • Development of biosensors based on ATP synthase rotation

• Methodological approaches:

  • Directed evolution to select for desired properties

  • Rational design based on structural knowledge

  • High-throughput screening of variant libraries

These approaches may lead to the development of biohybrid systems that harness the remarkable efficiency of ATP synthase for sustainable energy production or nanoscale mechanical devices.