Recombinant Rickettsia rickettsii ATP synthase subunit alpha (atpA), partial

What is the biochemical function of ATP synthase subunit alpha (atpA) in Rickettsia rickettsii?

ATP synthase subunit alpha is a critical component of the F1 sector of ATP synthase (EC 3.6.3.14), which is responsible for catalyzing ATP synthesis from ADP and inorganic phosphate. In Rickettsia rickettsii, this enzyme complex plays an essential role in energy metabolism, utilizing the proton gradient established across the bacterial membrane to drive ATP production. The alpha subunit specifically contributes to forming the catalytic sites within the F1 sector, working in concert with the beta subunit to bind substrates and facilitate the phosphorylation reaction. The recombinant partial form typically includes the catalytically important regions while excluding portions that might inhibit proper expression or folding . Understanding the function of atpA is fundamental to comprehending R. rickettsii energy metabolism and potentially identifying novel therapeutic targets against this pathogen.

What expression systems are typically used for producing recombinant R. rickettsii ATP synthase subunits?

Recombinant R. rickettsii ATP synthase subunit alpha (atpA) is typically expressed in mammalian cell systems as indicated in product specifications . This expression system is chosen for certain proteins where proper folding or post-translational modifications are critical for maintaining native structure and function. In contrast, other ATP synthase subunits from R. rickettsii, such as the delta subunit (atpH) and subunit a (atpB), have been successfully expressed in E. coli expression systems . The choice between mammalian and bacterial expression systems depends on multiple factors including protein complexity, disulfide bond requirements, glycosylation needs, and intended downstream applications. Expression in mammalian systems generally provides an environment more similar to the native eukaryotic host cells that R. rickettsii infects, potentially preserving important structural characteristics that might be lost in prokaryotic expression systems. Researchers should consider these factors when selecting an expression system for their specific research objectives.

How does atpA contribute to the pathogenesis of Rocky Mountain spotted fever?

R. rickettsii is the causative agent of Rocky Mountain spotted fever (RMSF), the most lethal tick-borne disease in the United States . While the search results don't explicitly connect atpA to pathogenesis mechanisms, energy metabolism is crucial for bacterial survival during infection. ATP synthase function is essential for maintaining adequate energy supplies during the intracellular life cycle of R. rickettsii, especially during active replication within host cells. The bacteria must generate ATP efficiently to support various energy-dependent processes including protein synthesis, DNA replication, and membrane transport mechanisms that facilitate host cell manipulation. The infection process involves complex host-pathogen interactions, with R. rickettsii actively replicating at tick bite sites and producing epidermal and dermal necrotic lesions characterized as inoculation eschars within days of infection . Understanding how atpA and other components of energy metabolism contribute to this process could reveal potential intervention points for therapeutic development.

What are the critical amino acid residues or domains in R. rickettsii atpA that affect function?

While the complete amino acid sequence of R. rickettsii atpA is not provided in the search results, functional domains can be inferred from homologous proteins. Typically, ATP synthase alpha subunits contain nucleotide-binding domains, catalytic residues involved in ATP synthesis, and interface regions that mediate interactions with other subunits of the ATP synthase complex. Critical domains likely include the Walker A and Walker B motifs that form the nucleotide-binding pocket, DELSEED sequence involved in conformational changes during catalysis, and residues that interact with the gamma subunit during rotational catalysis. Researchers studying the partial recombinant atpA should identify which specific domains are included in their construct to understand functional limitations. Sequence alignment with well-characterized ATP synthase alpha subunits from other organisms can help identify conserved and divergent regions that might influence function or serve as specific targets for antibody development . Site-directed mutagenesis of these conserved residues would provide experimental validation of their functional importance.

How does the regulation of atpA expression in R. rickettsii compare to the ATPA gene regulation in mammalian systems?

The regulation of ATP synthase genes differs substantially between prokaryotic and eukaryotic systems. In mammalian cells, the ATPA gene regulation involves complex mechanisms including transcription factors such as upstream stimulatory factor 2 (USF2) and YY1 that bind to initiator elements and E-box elements in the promoter region . These factors can either activate or repress transcription depending on cellular conditions. In contrast, bacterial gene regulation typically involves operons, with multiple related genes transcribed together from a single promoter. R. rickettsii, as an obligate intracellular bacterium with a reduced genome, likely has streamlined regulatory mechanisms adapted to its intracellular lifestyle. The expression of atpA in R. rickettsii is probably coordinated with other ATP synthase subunits and influenced by environmental factors such as pH, nutrient availability, and host cell interactions. Understanding these regulatory differences is important when studying recombinant protein expression and when considering how the ATP synthase complex might respond to different environmental conditions during infection .

What are the optimal storage and handling conditions for recombinant R. rickettsii atpA?

According to product specifications, recombinant R. rickettsii ATP synthase subunit alpha should be stored at -20°C, with extended storage at -20°C or -80°C . Working aliquots can be maintained at 4°C for up to one week, but repeated freezing and thawing should be avoided to prevent protein degradation and loss of activity. Prior to opening, vials should be briefly centrifuged to bring contents to the bottom. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL. Addition of glycerol to a final concentration of 5-50% is recommended for long-term storage, with 50% being the default recommendation . These storage conditions are designed to maintain protein stability and prevent degradation from proteases, oxidation, or aggregation. Researchers should carefully follow these guidelines to ensure the recombinant protein maintains its structural integrity and functional properties throughout their experimental timeline.

What purification methods are most effective for recombinant R. rickettsii atpA?

The recombinant R. rickettsii atpA is reported to have >85% purity as determined by SDS-PAGE , indicating effective purification methodology. While specific purification protocols aren't detailed in the search results, standard approaches for recombinant proteins from mammalian cell expression systems typically include initial clarification of cell lysates followed by chromatographic separation techniques. Common methods would include affinity chromatography (particularly if the recombinant protein contains an affinity tag), ion exchange chromatography based on the protein's isoelectric point, and size exclusion chromatography for final polishing and buffer exchange. The tag type for recombinant atpA is determined during the manufacturing process , and if a His-tag is used (as with the atpB protein in result ), immobilized metal affinity chromatography (IMAC) would be the primary purification method. Researchers may need to optimize purification conditions including buffer composition, pH, salt concentration, and elution gradients to maximize yield while maintaining protein activity and structural integrity.

What methods can be used to assess the functionality of recombinant R. rickettsii atpA?

Assessing the functionality of recombinant atpA presents unique challenges because it normally functions as part of the larger ATP synthase complex. Several complementary approaches can be employed to evaluate different aspects of its function. Binding assays using fluorescently labeled nucleotides can assess the protein's ability to bind ATP/ADP. Circular dichroism spectroscopy can verify proper secondary structure folding, while thermal shift assays can evaluate stability and potential ligand interactions. For more comprehensive functional assessment, reconstitution experiments with other ATP synthase subunits can test the ability of atpA to form proper subunit interactions and contribute to complex assembly. Additionally, antibody recognition using specific antibodies against conformational epitopes can indicate proper folding of critical domains. Enzymatic activity might be partially assessed through ATP hydrolysis assays, though full ATP synthesis activity would require reconstitution of the complete complex . These methodological approaches provide researchers with a toolkit for verifying that their recombinant protein preparation maintains the structural and functional characteristics necessary for meaningful experimental results.

How do the functional properties of recombinant atpA compare to those of other ATP synthase subunits from R. rickettsii?

The ATP synthase complex in R. rickettsii consists of multiple subunits with distinct functions that work cooperatively to generate ATP. The alpha subunit (atpA) is part of the F1 sector and contains nucleotide binding sites that participate in the catalytic mechanism of ATP synthesis . In comparison, the delta subunit (atpH) functions primarily as a connecting element between the F1 and F0 sectors, serving a structural role in complex assembly and stability . The a subunit (atpB) is a membrane-embedded component of the F0 sector that forms part of the proton channel, facilitating proton translocation across the membrane that drives ATP synthesis . The functional properties of these subunits reflect their distinct roles: atpA is involved in catalysis and energy transduction, atpH in structural organization, and atpB in proton transport. The amino acid sequences reflect these specialized functions, with atpA containing nucleotide-binding domains, atpH featuring domains for protein-protein interactions, and atpB having hydrophobic transmembrane regions (as evident in the provided sequence: MTHSPLAQFDIKKLIDIKMFGFDVSFTNSSIYMLLASILALTYFYLAFYKRKLVPSRLQV SAEIVYNLVADMLNQNIGVKGRKFIPLVFSLFIFILFCNLLGMTPYSFTATSHIIVTFTL AILVFLIVTIVGFVKHGLRFLTLFLPHGTPLWLAPLMIVIELFTYLARPVSLSLRLAANM MAGHVLLKVIAGFTVSLMIYLKFLPIPIMVILIGFEIFVAILQAYIFTILSCMYLNDAIN LH) .

What insights can comparative analysis of Rickettsia ATP synthase subunits provide for evolutionary studies?

Comparative analysis of ATP synthase subunits across Rickettsia species and other bacteria can reveal important evolutionary relationships and adaptation mechanisms. ATP synthase is a highly conserved enzyme complex essential for cellular energy production, making it an excellent subject for studying evolutionary processes. Analysis of sequence conservation, divergence, and selection pressures on different subunits can indicate which regions are functionally critical (highly conserved) versus those that may be involved in adaptation to specific hosts or environments (more variable). The alpha subunit (atpA) is generally more conserved than membrane-embedded subunits due to its central role in the catalytic mechanism. Comparison between R. rickettsii ATP synthase subunits and those from less pathogenic Rickettsia species could potentially identify sequence variations that correlate with virulence or host range . Additionally, since Rickettsia species are closely related to the ancestral endosymbiont that gave rise to mitochondria, comparative analysis between rickettsial and mitochondrial ATP synthase components provides insights into endosymbiotic evolution and the development of eukaryotic energy metabolism . These evolutionary studies can inform both basic science questions about bacterial adaptation and applied research into pathogenesis mechanisms.

How can recombinant R. rickettsii atpA be used for developing diagnostic tools for Rocky Mountain spotted fever?

Recombinant R. rickettsii atpA has significant potential for developing improved diagnostic tools for Rocky Mountain spotted fever (RMSF). The >85% purity recombinant protein could serve as an antigen for developing highly specific antibody-based detection systems. ELISA, immunofluorescence assays, or lateral flow immunochromatographic tests utilizing purified recombinant atpA could enable more specific detection of R. rickettsii antibodies in patient serum. This approach might help distinguish RMSF from other rickettsial diseases, addressing the diagnostic challenge noted in search result where "recent serological and epidemiological studies suggest that novel species of SFG Rickettsia are responsible for the increased number of cases of RMSF-like rickettsioses in the United States." Additionally, the recombinant protein could be used to generate high-quality antibodies for direct detection of the pathogen in clinical samples or tick vectors. PCR-based molecular diagnostics targeting the atpA gene could also be developed, with recombinant atpA serving as positive control material. These diagnostic applications would benefit from the specificity and reproducibility of the recombinant protein, potentially improving both the sensitivity and specificity of current diagnostic methods.

What structural approaches can elucidate the three-dimensional conformation of R. rickettsii atpA?

Elucidating the three-dimensional structure of R. rickettsii atpA would provide valuable insights into its function and potential as a therapeutic target. Several complementary structural biology approaches could be employed using the recombinant protein. X-ray crystallography would offer the highest resolution structural information, requiring crystallization of the purified recombinant protein followed by X-ray diffraction analysis. Cryo-electron microscopy (cryo-EM) provides an alternative approach that doesn't require crystallization, particularly valuable for membrane-associated proteins or larger complexes if atpA is studied in association with other ATP synthase components. Nuclear magnetic resonance (NMR) spectroscopy could provide information on protein dynamics and ligand interactions for smaller domains of the protein. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) could map surface-exposed regions and conformational changes upon nucleotide binding or interaction with other subunits. Computational approaches including homology modeling based on known structures of homologous proteins would complement experimental methods, particularly since ATP synthase is a well-studied enzyme complex in other organisms . The high purity (>85%) of the recombinant protein preparation makes it suitable for these structural studies, though optimization of buffer conditions might be necessary for specific techniques.

What are common challenges in expressing and purifying functional recombinant R. rickettsii atpA, and how can they be addressed?

Researchers working with recombinant R. rickettsii atpA may encounter several challenges during expression and purification. One common issue is low expression yield in mammalian cell systems , which might be addressed by optimizing expression conditions including cell line selection, transfection protocol, expression vector design, and culture conditions. Protein solubility can be problematic, especially if the recombinant construct includes hydrophobic regions; this may be improved by using solubility-enhancing tags, adjusting lysis buffer composition, or expressing only soluble domains. Maintaining proper protein folding is crucial, particularly for functional studies; careful selection of cell type and expression temperature, along with the addition of chaperones, may help. Protein stability during purification and storage presents another challenge, as indicated by the specific storage recommendations in the product information . This can be addressed through buffer optimization, addition of stabilizing agents like glycerol (recommended at 5-50%), and minimizing freeze-thaw cycles. Purification challenges include achieving high purity while maintaining activity; a multi-step purification strategy may be necessary, and inclusion of protease inhibitors throughout the process can prevent degradation. Validation of proper folding and function should include both structural assessments (e.g., circular dichroism) and functional assays appropriate to the intended research application.

How can researchers troubleshoot experiments involving R. rickettsii atpA when results deviate from expectations?

When experimental results with recombinant R. rickettsii atpA deviate from expectations, a systematic troubleshooting approach should be employed. First, researchers should verify protein quality by reassessing purity via SDS-PAGE (aiming for >85% as specified in the product information ), checking for degradation through Western blotting, and confirming protein concentration using multiple methods such as Bradford assay and absorbance at 280 nm. Storage conditions should be reviewed to ensure the protein hasn't been subjected to excessive freeze-thaw cycles or improper temperature storage, as this can significantly impact functionality. For binding or activity assays, buffer conditions including pH, ionic strength, and presence of divalent cations (particularly Mg2+) should be optimized, as these can dramatically affect ATP synthase subunit function. When antibody recognition is poor, epitope accessibility should be considered; mild denaturation or different fixation protocols might expose hidden epitopes. For structural studies, sample heterogeneity might be addressed through additional purification steps or screening different buffer conditions. When reconstituting with other subunits, stoichiometry and assembly order may need optimization. Control experiments using well-characterized homologous proteins from model organisms can provide valuable benchmarks. Documentation of all experimental conditions and systematic variation of parameters will facilitate identification of critical factors affecting experimental outcomes.