Recombinant Leptospira biflexa serovar Patoc ATP synthase subunit alpha (atpA), partial
Description
Protein Overview and Functional Significance
ATP synthase subunit alpha (atpA) is a core component of the F1Fo ATP synthase complex, responsible for ATP production in bacterial cells. In L. biflexa, this enzyme is essential for survival under varying environmental conditions. Key characteristics include:
The partial sequence suggests truncation for specific functional or structural studies, such as catalytic domain analysis or antibody production.
Genomic and Proteomic Context
The atpA gene is part of the L. biflexa genome, which comprises two chromosomes (CI and CII) and a third replicon (p74) . Comparative genomics reveals that L. biflexa retains metabolic genes shared with pathogenic Leptospira species but lacks virulence-associated loci. Key findings:
-
Gene Density: L. biflexa’s genome has high gene density, limiting large-scale rearrangements .
-
Posttranslational Modifications: ATP synthase subunits in L. biflexa may undergo methylation, acetylation, or phosphorylation, as observed in proteomic studies .
-
Evolutionary Conservation: ATP synthase components are conserved across Leptospira species, making atpA a target for cross-species metabolic studies .
Heterologous Expression and Research Applications
While no direct studies on recombinant atpA were identified, L. biflexa is widely used as a model for heterologous expression of pathogenic Leptospira genes due to its genetic tractability . For example:
-
Expression Systems: The pMaOri vector and lipL32 promoter (P32) have been used to overexpress foreign proteins in L. biflexa with high efficiency .
-
Functional Studies: Recombinant proteins like LIC11711 (adhesion factor) and LMB26 (fibronectin-binding protein) have been expressed in L. biflexa to study host-pathogen interactions .
A hypothetical workflow for recombinant atpA production could involve:
-
Cloning: Amplification of the partial atpA sequence and insertion into a shuttle vector (e.g., pMaOri).
-
Transformation: Introduction into L. biflexa or E. coli for expression.
-
Purification: Affinity chromatography using tags like His6, followed by SDS-PAGE validation .
Potential Research Implications
-
Vaccine Development: Recombinant atpA could serve as an antigen for cross-protective immunity studies, similar to L. biflexa’s use in ELISA diagnostics .
-
Metabolic Engineering: Disruption or overexpression of atpA might elucidate ATP synthase’s role in Leptospira survival under nutrient-limited conditions .
-
Structural Biology: Partial sequences allow crystallography or cryo-EM studies of specific ATP synthase domains.
Limitations and Knowledge Gaps
-
No direct experimental data on recombinant atpA from L. biflexa were found in the reviewed literature.
-
Functional assays (e.g., ATP hydrolysis activity) and immunogenicity studies remain areas for future investigation.
Product Specs
Target Background
KEGG: lbf:LBF_0776
Q&A
Basic Research Questions
What is the biological role of ATP synthase subunit alpha (AtpA) in Leptospira biflexa metabolism?
ATP synthase subunit alpha (AtpA) is a critical component of the F domain of ATP synthase, which catalyzes ATP synthesis via oxidative phosphorylation. In L. biflexa, AtpA facilitates the conversion of ADP to ATP by coupling proton translocation across the membrane to mechanical rotation of the enzyme’s γ-subunit . Methodological validation involves:
-
Functional assays: Measuring ATP hydrolysis/synthesis rates in membrane fractions using luciferase-based luminescence or spectrophotometric phosphate detection .
-
Gene silencing: CRISPR interference (CRISPRi) to knock down atpA expression and observe metabolic disruption (e.g., growth arrest in EMJH medium lacking iron) .
-
Structural analysis: Cryo-EM or X-ray crystallography to resolve rotational mechanics of the FF complex .
How is recombinant AtpA from L. biflexa serovar Patoc produced and purified?
Recombinant AtpA is typically expressed in heterologous systems such as E. coli due to L. biflexa’s fastidious growth requirements. Key steps include:
-
Cloning: Amplify atpA from L. biflexa genomic DNA (strain Patoc 1) using primers flanking the partial sequence (e.g., residues 1–294). Ligate into IPTG-inducible vectors (e.g., pMaOri) with a His-tag for affinity purification .
-
Expression: Optimize induction with 1 mM IPTG for 24 hours at 30°C .
-
Purification: Use nickel-NTA chromatography under denaturing conditions (8 M urea) if inclusion bodies form, followed by refolding via dialysis . Validate purity via SDS-PAGE (>90%) and immunoblotting with anti-AtpA antibodies .
What methods validate the structural integrity of recombinant AtpA?
-
Circular dichroism (CD): Confirm secondary structure composition (e.g., α-helix dominance) by comparing spectra to wild-type AtpA .
-
Size-exclusion chromatography (SEC): Assess oligomeric state; native AtpA forms part of a 550 kDa FF complex .
-
ATPase activity assays: Compare recombinant and native enzyme kinetics (e.g., for ATP hydrolysis) to confirm functional fidelity .
Advanced Research Questions
How can contradictions in ATP synthase functional assays across studies be resolved?
Discrepancies often arise from differences in:
-
Experimental systems: In vitro reconstitutions vs. whole-cell assays (e.g., proteoliposomes vs. live L. biflexa ).
-
Posttranslational modifications (PTMs): L. biflexa AtpA exhibits methylation and acetylation, altering enzymatic activity . Use PTM-specific antibodies or mass spectrometry to quantify modifications (Table 1).
-
Proton motive force (PMF) conditions: Standardize PMF (e.g., ΔpH and ΔΨ) using ionophores (e.g., nigericin) in proteoliposome assays .
Table 1: Common PTMs in L. biflexa AtpA and Functional Impact
What experimental designs study in vivo interactions of AtpA with other ATP synthase subunits?
-
Bacterial two-hybrid (BTH): Clone atpA and partner subunits (e.g., β, γ) into pKT25/pUT18C vectors. Measure β-galactosidase activity in E. coli to map interactions .
-
Crosslinking mass spectrometry (XL-MS): Treat L. biflexa membranes with DSS (disuccinimidyl suberate), immunoprecipitate AtpA, and identify bound subunits via LC-MS/MS .
-
CRISPRi-based knockdown: Silencing atpB (subunit beta) and assessing AtpA stability via pulse-chase assays .
How can structural data from homologous ATP synthases improve L. biflexa AtpA modeling?
-
Homology modeling: Use templates like E. coli AtpA (PDB: 1FS0) or S. cerevisiae F-ATPase (PDB: 2HLD) in SWISS-MODEL. Validate with MD simulations (e.g., GROMACS) to assess rotor-stator dynamics .
-
SAXS: Compare low-resolution solution structures of recombinant AtpA with full-length F complexes to identify domain flexibility .
-
Electron cryotomography (cryo-ET): Image native L. biflexa membranes to resolve subunit spatial organization .
Methodological Challenges and Solutions
Why do recombinant AtpA constructs often lack enzymatic activity, and how is this addressed?
-
Cause: Improper folding due to missing subunits (e.g., β or γ) or absence of membrane-embedded F domain .
-
Solutions:
How to reconcile low yields of recombinant AtpA with high experimental demand?
-
Optimize codon usage: Redesign atpA with E. coli-preferred codons via tools like GeneOptimizer .
-
Switch expression systems: Use Pichia pastoris for eukaryotic PTMs or in vitro translation systems for toxic constructs .
-
Scale-up fermentation: Employ high-density bioreactors with controlled pH (7.2) and dissolved oxygen (>30%) .
Data Interpretation Guidelines
-
Unexpected ATP synthesis inhibition: Check for contaminating ADP/ATPases via enzymatic assays with/without inhibitors (e.g., oligomycin) .
-
Discrepant PTM profiles: Compare L. biflexa growth phases (exponential vs. stationary); methylation peaks in late log phase .
-
Non-reproducible structural data: Standardize buffer conditions (e.g., 100 mM glycine, pH 9.5) to match Leptospira membrane physiology .
