Recombinant Tropheryma whipplei ATP synthase subunit c (atpE)

What is the basic structure and function of Tropheryma whipplei ATP synthase subunit c?

Tropheryma whipplei ATP synthase subunit c (atpE) is a critical component of the F-type ATP synthase complex in this bacterium. The protein consists of 75 amino acids with the sequence: MGSVLAEVAGSLASIGYGLAAIGSAIGVGIVVGKTVESVARQPELAKRLTVLMYVGVAFTEALALIGIGTYFLFR . Functionally, it serves as a key component of the F0 sector of ATP synthase, forming part of the c-ring structure that facilitates proton translocation across the membrane. Similar to other F-type ATPases, the c-subunit is involved in the rotary mechanism that couples proton movement to ATP synthesis or hydrolysis . The protein is predominantly hydrophobic, containing transmembrane α-helices that span the membrane and contribute to the formation of the proton channel.

How does T. whipplei ATP synthase subunit c compare structurally to ATP synthase components in other organisms?

The c-subunit of T. whipplei forms part of the membrane-embedded rotor that, in conjunction with the F1 subcomplex, establishes the complete ATP synthase machinery. Research has demonstrated that the F1 subcomplex acts as a gate for the c-subunit pore, regulating channel activity and potentially participating in mitochondrial permeability transition mechanisms .

What are the established protocols for expression and purification of recombinant T. whipplei atpE?

For researchers seeking to express and purify recombinant T. whipplei ATP synthase subunit c, both prokaryotic and eukaryotic expression systems have been successfully employed. The protein can be produced with appropriate tags to facilitate purification, though the specific tag should be determined during the production process based on experimental needs .

Methodology:

• Clone the atpE gene (TW339 locus) into an appropriate expression vector

• Transform into expression host (E. coli for prokaryotic expression or suitable eukaryotic system)

• Induce protein expression under optimized conditions

• Lyse cells and perform initial purification steps

• Utilize affinity chromatography based on the selected tag

• Perform size exclusion or ion exchange chromatography for further purification

• Store in Tris-based buffer with 50% glycerol at -20°C for short-term or -80°C for long-term storage

For optimal stability, avoid repeated freeze-thaw cycles and store working aliquots at 4°C for up to one week .

What methods are most effective for detecting T. whipplei ATP synthase in clinical and research samples?

Detection of T. whipplei ATP synthase components in clinical and research samples can be achieved through several methods, with immunological techniques showing particular promise. Monoclonal antibodies (MAbs) directed against T. whipplei ATP synthase F1 complex beta chain (58-kDa protein) have demonstrated high specificity and sensitivity .

Recommended detection methods:

• Immunofluorescence assay (IFA): Monoclonal antibodies against the ATP synthase F1 complex beta chain have shown excellent specificity for T. whipplei detection in various samples, including stool, with minimal background compared to polyclonal antibodies .

• Western blotting: Using MAbs specific to the 58-kDa ATP synthase F1 complex beta chain allows specific detection of T. whipplei proteins in complex mixtures. Two-dimensional gel electrophoresis combined with western blotting can further improve specificity .

• Mass spectrometry: For definitive identification, trypsin in-gel digestion followed by mass spectrometry with matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) has successfully identified the ATP synthase components of T. whipplei .

These methods have been validated across multiple strains of T. whipplei, demonstrating their reliability for both research and potentially diagnostic applications.

What are the challenges in generating functional recombinant ATP synthase subunit c, and how can they be overcome?

Generating functional recombinant ATP synthase subunit c presents several challenges due to its hydrophobic nature and role in multiprotein complexes. Researchers should consider the following methodological approaches:

Challenges and solutions:

• Protein solubility: The hydrophobic nature of subunit c often leads to aggregation during expression and purification.

  • Solution: Utilize detergent-based extraction methods or fusion partners that enhance solubility, such as maltose-binding protein.

• Proper folding: Ensuring correct protein folding in recombinant systems.

  • Solution: Consider expression at lower temperatures (16-25°C) and use of molecular chaperones.

• Functional assessment: Determining if the recombinant protein retains native activity.

  • Solution: Reconstitution experiments in liposomes to assess proton conductance, or association studies with other ATP synthase components.

• Storage stability: Maintaining protein integrity during storage.

  • Solution: Store in glycerol-containing buffers (50%) at -20°C or -80°C and avoid repeated freezing and thawing cycles .

How can researchers effectively analyze the interaction between T. whipplei ATP synthase subunit c and other components of the ATP synthase complex?

Understanding the interactions between T. whipplei ATP synthase subunit c and other complex components requires sophisticated biochemical and biophysical approaches:

Methodological approaches:

• Co-immunoprecipitation: Using antibodies against the c-subunit or other components to pull down interaction partners.

• Surface plasmon resonance (SPR): For quantitative assessment of binding kinetics between purified components.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry analysis to identify interaction points.

• Functional reconstitution: Reconstituting purified components into liposomes to assess functional interactions, particularly focusing on how the F1 subcomplex interacts with and regulates the c-subunit channel activity .

• Structural biology approaches: Cryo-electron microscopy or X-ray crystallography of the assembled complex or subcomplexes to define atomic-level interactions.

Evidence suggests that the F1 subcomplex plays a crucial role in gating the c-subunit pore, affecting channel activity in a regulatory manner that may be particularly important during cellular stress responses .

How does the ATP synthase subunit c contribute to T. whipplei pathogenesis in Whipple's disease?

The ATP synthase subunit c of T. whipplei likely plays multiple roles in bacterial pathogenesis and the development of Whipple's disease:

• Energy metabolism: As a component of ATP synthase, it plays a fundamental role in bacterial energy production, supporting survival and replication within host cells.

• Antigenic properties: Research has demonstrated that ATP synthase components of T. whipplei are immunogenic, with the F1 complex beta chain (58-kDa) identified as an immunodominant epitope recognized by multiple monoclonal antibodies . This immunogenicity may contribute to the host immune response during infection.

• Potential diagnostic marker: The ATP synthase components, particularly the F1 complex beta chain, have been identified as specific targets for antibody-based detection methods, suggesting their potential value in diagnostic applications for Whipple's disease .

• Target for therapeutic intervention: Understanding the structure and function of the ATP synthase components may reveal potential targets for antimicrobial development specific to T. whipplei.

While direct evidence linking the c-subunit to specific pathogenicity mechanisms is limited, its essential role in bacterial energy metabolism makes it inherently important for bacterial survival and therefore disease progression.

What is the potential of T. whipplei ATP synthase components as diagnostic markers for Whipple's disease?

T. whipplei ATP synthase components show significant promise as diagnostic markers for Whipple's disease, based on several experimental findings:

• Specificity: Monoclonal antibodies directed against the ATP synthase F1 complex beta chain of T. whipplei have demonstrated high specificity, showing no cross-reactivity with 22 different bacterial species tested, including other actinobacteria .

• Strain independence: Despite the specificity for T. whipplei, these antibodies recognize multiple strains of the bacterium, indicating they target conserved epitopes across different T. whipplei isolates .

• Detection in clinical samples: Preliminary studies have shown that monoclonal antibodies against T. whipplei ATP synthase components can effectively detect the bacterium in stool samples with lower background fluorescence compared to polyclonal antibodies .

• Mass spectrometry identification: The ATP synthase components can be reliably identified by mass spectrometry following two-dimensional gel electrophoresis, providing a confirmatory method for detection .

Potential diagnostic applications:

• Immunofluorescence assays for detecting T. whipplei in tissue biopsies or stool samples

• ELISA-based detection systems using recombinant ATP synthase components

• Mass spectrometry-based identification in complex clinical samples

Further validation in larger clinical cohorts would be necessary to establish the sensitivity and specificity of these approaches for routine diagnostic use.

How can structural studies of T. whipplei ATP synthase c-subunit inform drug discovery efforts for Whipple's disease?

Structural studies of the T. whipplei ATP synthase c-subunit could significantly advance drug discovery efforts through several approaches:

• Structure-based drug design: Detailed structural information about the c-subunit, particularly its unique features compared to human homologs, could enable the rational design of small molecules that specifically target T. whipplei ATP synthase without affecting host enzymes.

• Identification of functional hotspots: Structural analysis can reveal critical regions for protein function that might serve as vulnerable targets for therapeutic intervention.

• Understanding assembly mechanisms: Structural insights into how the c-subunit interacts with other components of the ATP synthase complex could reveal potential sites for disrupting complex assembly, thereby inhibiting bacterial energy production.

• Comparative structural biology: Comparing the structure of T. whipplei ATP synthase components with those from other bacteria and humans could identify unique structural features that might be exploited for selective targeting.

• Allosteric site identification: Beyond the active site, structural studies might reveal allosteric sites that could be targeted to modulate enzyme function in a highly specific manner.

The methodological approach would involve X-ray crystallography or cryo-electron microscopy of the purified c-subunit, ideally in different conformational states or in complex with other ATP synthase components, to provide a comprehensive structural understanding that can guide therapeutic development.

What is known about the regulatory mechanisms controlling T. whipplei ATP synthase activity, and how might these be experimentally investigated?

Our understanding of the regulatory mechanisms controlling T. whipplei ATP synthase activity is still developing, but several approaches can be used to investigate these mechanisms:

Current knowledge and investigative approaches:

• F1 subcomplex as a gate: Research on ATP synthase components suggests that the F1 subcomplex functions as a gate for the c-subunit pore, regulating channel activity . This regulatory mechanism could be investigated by:

  • Reconstitution experiments with and without the F1 subcomplex

  • Electrophysiological studies measuring channel activity under different conditions

  • Site-directed mutagenesis of potential regulatory sites

• Role in mitochondrial permeability transition: ATP synthase c-ring has been implicated in forming the leak channel of mitochondrial permeability transition (mPT), activated during cellular stress . This could be investigated in T. whipplei by:

  • Calcium sensitivity assays

  • Membrane potential measurements

  • Comparative studies with other bacterial ATP synthases

• Response to environmental conditions: How T. whipplei ATP synthase activity responds to changes in pH, ion concentrations, and energy status could be studied through:

  • Enzyme activity assays under varying conditions

  • Gene expression analysis in different environments

  • Protein modification (phosphorylation, etc.) studies

These investigations would contribute to our understanding of T. whipplei energy metabolism and potentially reveal new targets for therapeutic intervention.

What are the emerging techniques for studying ATP synthase function in T. whipplei and related bacteria?

Several cutting-edge techniques are emerging for the study of ATP synthase function in bacteria like T. whipplei:

• Single-molecule techniques:

  • Single-molecule FRET (Förster Resonance Energy Transfer) to monitor conformational changes

  • Optical tweezers to measure force generation and mechanical properties

  • High-speed AFM (Atomic Force Microscopy) to visualize rotary motion in real-time

• Advanced structural methods:

  • Cryo-electron tomography to visualize ATP synthase in its native membrane environment

  • Time-resolved X-ray crystallography to capture different functional states

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic regions and interactions

• Innovative functional assays:

  • Fluorescent probes to monitor proton pumping in real-time

  • Nanodiscs or other membrane mimetics for functional reconstitution

  • Microfluidic systems for high-throughput functional analysis

• Genetic and genomic approaches:

  • CRISPR-based genome editing for functional studies

  • Transcriptomic and proteomic analysis to understand regulation

  • Comparative genomics across T. whipplei strains to identify conserved features

These techniques, used in combination, could provide unprecedented insights into the structure, function, and regulation of T. whipplei ATP synthase, potentially revealing new targets for therapeutic intervention in Whipple's disease.

How does the ATP synthase c-subunit from T. whipplei compare with homologous proteins in other bacterial pathogens?

Comparative analysis reveals both similarities and distinctions between the ATP synthase c-subunit of T. whipplei and those of other bacterial pathogens:

Structural and functional comparisons:

Future comparative studies utilizing structural biology approaches and functional assays across different bacterial pathogens could further elucidate the unique features of the T. whipplei ATP synthase c-subunit.

What are promising future research directions for understanding the role of ATP synthase in T. whipplei biology and pathogenesis?

Several promising research directions could advance our understanding of ATP synthase's role in T. whipplei biology and pathogenesis:

• Host-pathogen interactions:

  • Investigate how T. whipplei ATP synthase components interact with host immune system components

  • Determine if ATP synthase subunits are exposed on the bacterial surface and potentially interact with host receptors

  • Explore whether host factors directly modulate T. whipplei ATP synthase activity

• Drug development:

  • Screen for small molecules that specifically inhibit T. whipplei ATP synthase

  • Develop peptide-based inhibitors targeting unique regions of the ATP synthase complex

  • Explore combination therapies targeting ATP synthase along with other essential bacterial processes

• System-level understanding:

  • Map the metabolic network connected to ATP synthase function in T. whipplei

  • Investigate how energy metabolism adapts during different phases of infection

  • Develop mathematical models of T. whipplei bioenergetics

• Diagnostic applications:

  • Develop improved antibody-based detection methods targeting ATP synthase components

  • Explore mass spectrometry-based diagnostics for direct detection in clinical samples

  • Investigate serological responses to ATP synthase components as diagnostic markers

These research directions would not only advance our fundamental understanding of T. whipplei biology but could also lead to practical applications in diagnosis and treatment of Whipple's disease.