Recombinant Citrobacter koseri ATP synthase subunit a (atpB)

How can I express and purify recombinant C. koseri ATP synthase subunit a?

For expression and purification of recombinant C. koseri ATP synthase subunit a, the following methodological approach is recommended:

• Gene cloning: Amplify the atpB gene from C. koseri genomic DNA using PCR with high-fidelity polymerase and sequence-specific primers.

• Vector construction: Clone the amplified gene into an expression vector containing a suitable tag (His-tag is commonly used for membrane proteins) for purification.

• Expression system: Transform the construct into an appropriate E. coli strain optimized for membrane protein expression (e.g., C41(DE3) or C43(DE3)).

• Culture conditions: Grow at lower temperatures (16-25°C) after induction to enhance proper folding.

• Membrane isolation: Disrupt cells via sonication or French press, followed by differential centrifugation to isolate membrane fractions.

• Solubilization: Use mild detergents (e.g., n-dodecyl-β-D-maltoside or digitonin) to extract the membrane protein.

• Purification: Employ affinity chromatography using the tag, followed by size exclusion chromatography for higher purity.

What are the common challenges when working with recombinant C. koseri atpB?

Working with recombinant C. koseri atpB presents several methodological challenges:

• Membrane protein solubility: As a membrane-embedded protein, atpB tends to form inclusion bodies or aggregate when overexpressed.

• Protein misfolding: The hydrophobic nature of membrane proteins often leads to improper folding in recombinant expression systems.

• Low yield: Membrane proteins typically express at lower levels compared to soluble proteins.

• Functional assessment: Traditional enzymatic assays may be challenging as the protein functions as part of a multi-subunit complex.

• Structural integrity: Maintaining the native conformation during purification requires careful selection of detergents and buffer conditions.

To address these challenges, consider using specialized expression hosts, fusion partners that enhance solubility, lower induction temperatures, and optimized detergent screens for purification.

How does C. koseri ATP synthase compare to other bacterial ATP synthases?

While C. koseri ATP synthase shares fundamental structural and functional properties with other bacterial ATP synthases, its specific characteristics make it worthy of focused research:

The C. koseri ATP synthase has been studied less extensively than its E. coli counterpart, making it a valuable research target, particularly in the context of opportunistic infections in immunocompromised patients .

What are the current approaches for targeting C. koseri ATP synthase for antimicrobial development?

Research on C. koseri ATP synthase as an antimicrobial target has employed several sophisticated approaches:

• Structure-based drug design: Using the predicted 3D structure of C. koseri ATP synthase from SWISS-MODEL and AlphaFold models to identify potential binding pockets for inhibitor design .

• Ligand-based pharmacophore modeling: Developing pharmacophore models based on known ATP synthase inhibitors such as ampicillin to identify chemical features critical for binding .

• Virtual screening and molecular docking: Large-scale screening of compound libraries against the ATP synthase structure, with the top compounds selected based on binding affinities. Recent research identified compounds with binding affinities ranging from -10.021 to -8.452 kcal/mol .

• ADMET profiling: Evaluating pharmacokinetic properties of potential inhibitors to prioritize compounds with favorable drug-like characteristics.

• Molecular dynamics simulations: Assessing the stability of protein-ligand complexes over time to confirm binding modes and identify stable interactions.

The most promising recent candidates include PubChem-25230613, PubChem-74936833, CHEMBL263035, and PubChem-44208924, which demonstrated favorable binding characteristics and ADMET profiles in computational studies .

How can I assess the functional integrity of recombinant C. koseri atpB in vitro?

Evaluating the functional integrity of recombinant C. koseri atpB requires specialized approaches:

• Reconstitution assays: Incorporate purified atpB into liposomes or nanodiscs along with other ATP synthase subunits to reconstitute functional complexes.

• Proton translocation measurements: Use pH-sensitive fluorescent dyes (e.g., ACMA or pyranine) to monitor proton movement across membranes containing reconstituted atpB.

• ATP synthesis activity: Measure ATP production in reconstituted systems using luciferase-based assays when a proton gradient is applied.

• Binding studies: Assess interaction with known ATP synthase inhibitors using techniques such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR).

• Structural integrity verification: Employ circular dichroism (CD) spectroscopy to confirm secondary structure content and thermal stability.

• Site-directed mutagenesis: Introduce mutations in conserved residues predicted to be essential for function and assess the impact on activity.

What role does atpB play in C. koseri virulence and opportunistic infections?

The role of ATP synthase subunit a in C. koseri virulence is a complex area of research with several important considerations:

• Energy provision for virulence: ATP synthase supplies the energy required for numerous virulence mechanisms, including type III secretion systems, flagellar motility, and toxin production.

• Adaptation to host environments: ATP synthase enables metabolic flexibility, allowing C. koseri to adapt to varying nutrient and oxygen conditions within the host.

• Biofilm formation: ATP production supports the energy-intensive process of biofilm development, which enhances persistence in hostile environments.

• Connection to immunocompromised hosts: C. koseri predominantly causes infections in individuals with compromised immunity or significant comorbidities, suggesting that under normal immune surveillance, the organism's virulence mechanisms (including ATP-dependent processes) are controlled .

• Link to underlying pathologies: There is evidence that C. koseri infections may be associated with underlying conditions such as malignancy, as demonstrated in a case where C. koseri pneumonia led to the discovery of pulmonary adenocarcinoma .

Recent studies have emphasized the importance of ATP synthesis in opportunistic pathogens like C. koseri, particularly in the context of emerging antibiotic resistance .

How can I design experiments to investigate atpB's role in antibiotic resistance mechanisms in C. koseri?

To investigate the role of ATP synthase subunit a in antibiotic resistance, consider the following experimental design approaches:

• Gene knockout/knockdown studies:

  • Create atpB deletion or conditional expression mutants

  • Compare minimum inhibitory concentrations (MICs) of various antibiotics between wild-type and mutant strains

  • Assess growth rates and fitness costs of mutations

• Overexpression analysis:

  • Express wild-type or mutated atpB variants in C. koseri

  • Evaluate changes in antibiotic susceptibility patterns

  • Measure energy production capacity in these strains

• Combination therapy assessment:

  • Test ATP synthase inhibitors in combination with conventional antibiotics

  • Determine synergistic, additive, or antagonistic effects using checkerboard assays

  • Calculate fractional inhibitory concentration indices (FICI)

• Resistance development monitoring:

  • Conduct serial passage experiments in sub-inhibitory concentrations of ATP synthase inhibitors

  • Sequence atpB and other ATP synthase genes to identify emerging mutations

  • Characterize cross-resistance patterns to different antibiotic classes

• Molecular basis investigation:

  • Identify blaKPC-containing C. koseri strains, which have been reported in previous research

  • Investigate potential metabolic adaptations in these resistant strains

  • Assess whether changes in ATP synthase expression or activity correlate with resistance profiles

What are the latest computational approaches for studying atpB structure-function relationships?

Advanced computational methods have become essential for studying structure-function relationships in proteins like C. koseri atpB:

• Homology modeling and ab initio structure prediction:

  • SWISS-MODEL has been used with E. coli ATP synthase (PDB ID: 6OQR) as a template

  • AlphaFold has provided ab initio models of C. koseri ATP synthase

  • Structure validation using tools like VERIFY, ERRAT, and MolProbity ensures model quality

• Molecular dynamics simulations:

  • All-atom simulations to analyze protein stability and conformational changes

  • Assessment of protein-membrane interactions in lipid bilayer environments

  • Analysis of proton translocation pathways through the membrane domain

• Quantum mechanics/molecular mechanics (QM/MM):

  • Hybrid methods to study proton transfer mechanisms with quantum-level accuracy

  • Investigation of catalytic residues involved in proton translocation

• Machine learning approaches:

  • Prediction of functional sites based on sequence conservation and structural features

  • Identification of potential drug binding sites through deep learning algorithms

• Virtual screening and docking:

  • Large-scale screening of compound libraries against predicted binding pockets

  • Recent studies screened 2043 compounds, with the top hits showing binding affinities between -10.021 and -8.452 kcal/mol

How can I design a recombinant expression system optimized specifically for C. koseri atpB?

Designing an optimized expression system for C. koseri atpB requires careful consideration of multiple factors:

• Codon optimization:

  • Analyze the codon usage bias in the target expression host

  • Optimize the atpB gene sequence to match the preferred codons without altering the amino acid sequence

  • Consider GC content and potential secondary structures in mRNA

• Expression vector selection:

  • Choose vectors with tunable promoters (e.g., rhamnose or tetracycline-inducible) for precise control

  • Consider fusion partners that enhance membrane protein expression (e.g., Mistic, SUMO, or GFP)

  • Incorporate affinity tags that minimally interfere with protein function

• Host strain engineering:

  • Select strains designed for membrane protein expression (C41/C43(DE3), Lemo21(DE3))

  • Consider knockout strains lacking proteases that degrade membrane proteins

  • Evaluate co-expression of chaperones (GroEL/GroES, DnaK/DnaJ) to assist folding

• Culture conditions optimization:

  • Test expression at various temperatures (16-30°C)

  • Evaluate different induction strategies (OD₆₀₀ at induction, inducer concentration)

  • Consider specialized media formulations to enhance membrane protein expression

• High-throughput screening:

  • Design a parallel expression screening approach with different combinations of vectors, hosts, and conditions

  • Use GFP fusion to rapidly assess proper folding and membrane insertion

  • Implement small-scale purification protocols to identify optimal conditions before scaling up

What techniques are most effective for studying the interaction between atpB and potential inhibitor compounds?

To effectively study interactions between C. koseri atpB and potential inhibitors, employ these methodological approaches:

• Biophysical techniques:

  • Isothermal Titration Calorimetry (ITC): Provides binding constants, stoichiometry, and thermodynamic parameters

  • Surface Plasmon Resonance (SPR): Enables real-time monitoring of binding kinetics

  • Microscale Thermophoresis (MST): Detects interactions with minimal protein consumption

  • Thermal Shift Assays: Monitors protein stabilization upon inhibitor binding

• Structural methods:

  • X-ray crystallography of atpB-inhibitor complexes (challenging but highly informative)

  • Cryo-electron microscopy (cryo-EM) of ATP synthase complexes with bound inhibitors

  • NMR spectroscopy for analyzing inhibitor binding sites and conformational changes

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions protected upon inhibitor binding

• Functional assays:

  • ATP synthesis inhibition assays using reconstituted systems

  • Proton translocation measurements in the presence of inhibitors

  • Growth inhibition assays with C. koseri cultures

• Computational approaches:

  • Molecular docking to predict binding modes

  • Molecular dynamics simulations to assess stability of protein-inhibitor complexes

  • Free energy calculations to estimate binding affinities

Recent computational screening identified compounds with binding affinities ranging from -10.021 to -8.452 kcal/mol, with the top four compounds (PubChem-25230613, PubChem-74936833, CHEMBL263035, PubChem-44208924) showing promising ADMET profiles and stable binding interactions .

How should I interpret discrepancies between computational models and experimental data for C. koseri atpB?

When confronted with discrepancies between computational predictions and experimental results for C. koseri atpB, consider these analytical approaches:

• Model validation and refinement:

  • Reassess the quality of the computational model using multiple validation metrics

  • Consider the limitations of template-based modeling, especially if sequence identity with the template is <30%

  • Refine models using experimental constraints from techniques like cross-linking or HDX-MS

• Experimental limitations analysis:

  • Evaluate whether the recombinant protein maintains native folding and function

  • Consider if detergents or reconstitution systems might alter protein behavior

  • Assess the sensitivity and specificity of experimental assays

• Contextual differences:

  • Computational models often represent static structures, while proteins are dynamic

  • Environmental factors (pH, ionic strength, membrane composition) may differ between computational assumptions and experimental conditions

  • Interactions with other ATP synthase subunits may influence atpB behavior

• Systematic comparison approach:

  • Create a detailed comparison table highlighting specific discrepancies

  • Prioritize investigation of critical functional residues or regions

  • Design targeted experiments to specifically address the discrepancies

• Integrated analysis:

  • Combine multiple computational approaches (homology modeling, molecular dynamics, QM/MM)

  • Triangulate with diverse experimental techniques

  • Consider ensemble-based approaches rather than single static models

What statistical approaches are appropriate for analyzing structure-activity relationships of potential atpB inhibitors?

For robust structure-activity relationship (SAR) analysis of potential C. koseri atpB inhibitors, these statistical approaches are recommended:

• Correlation analysis:

  • Pearson or Spearman correlation between structural descriptors and biological activity

  • Multiple linear regression (MLR) to identify key structural features influencing activity

  • Principal component analysis (PCA) to reduce dimensionality and identify patterns

• Quantitative Structure-Activity Relationship (QSAR) modeling:

  • Partial least squares (PLS) regression for handling many potentially correlated variables

  • Support vector machines (SVM) for classification of active versus inactive compounds

  • Random forest algorithms for handling complex, non-linear relationships

• Pharmacophore modeling:

  • Statistical validation of pharmacophore hypotheses using ROC curves and enrichment factors

  • Bootstrap analysis to assess model robustness

  • Cross-validation to evaluate predictive performance

• Machine learning approaches:

  • Neural networks for identifying complex patterns in structure-activity data

  • Bayesian models for activity prediction with uncertainty quantification

  • Transfer learning when working with limited datasets

• Model validation:

  • k-fold cross-validation to assess generalizability

  • External validation sets to evaluate predictive power

  • Y-scrambling to detect chance correlations

Recent computational studies applied these approaches to identify potential ATP synthase inhibitors with binding affinities ranging from -10.021 to -8.452 kcal/mol, demonstrating the utility of statistical methods in this research area .

What are emerging approaches for targeting ATP synthase in antibiotic-resistant C. koseri strains?

Several innovative approaches show promise for targeting ATP synthase in antibiotic-resistant C. koseri:

• Allosteric inhibitor development:

  • Targeting non-conserved allosteric sites specific to bacterial ATP synthases

  • Designing inhibitors that selectively disrupt conformational changes rather than directly blocking the active site

• Combination therapies:

  • Pairing ATP synthase inhibitors with conventional antibiotics to overcome resistance

  • Developing dual-action compounds that simultaneously target ATP synthase and other essential processes

• Immunomodulatory approaches:

  • Designing agents that both inhibit ATP synthase and enhance host immune response

  • Developing antibody-drug conjugates targeting bacterial ATP synthase

• Nanoparticle delivery systems:

  • Engineering nanoparticles for targeted delivery of ATP synthase inhibitors to infection sites

  • Developing pH-responsive nanocarriers that release inhibitors in the acidic microenvironment of bacterial infections

• CRISPR-Cas antimicrobials:

  • Designing CRISPR-Cas systems targeting atpB genes in resistant strains

  • Developing RNA-guided nucleases specific for resistance-associated mutations

The emergence of blaKPC-containing C. koseri strains highlights the urgent need for new antimicrobial approaches that can overcome existing resistance mechanisms .

How might research on C. koseri atpB contribute to our understanding of pathogenesis in immunocompromised hosts?

Research on C. koseri ATP synthase subunit a has significant implications for understanding opportunistic infections in immunocompromised hosts:

• Energy metabolism-virulence connections:

  • Investigating how ATP production supports various virulence mechanisms

  • Understanding metabolic adaptations that occur during transition from commensal to pathogen

• Host-pathogen interactions:

  • Exploring how ATP synthase activity influences bacterial persistence in immunocompromised environments

  • Investigating potential interactions between bacterial ATP synthase and host immune components

• Biomarker development:

  • Assessing whether ATP synthase components could serve as biomarkers for early detection of infection

  • Investigating atpB expression patterns during different stages of infection

• Personalized treatment approaches:

  • Developing therapeutic strategies targeting ATP synthase that account for specific immune deficiencies

  • Investigating how underlying conditions (such as malignancy) influence C. koseri infections and ATP synthase function

• Model system development:

  • Establishing C. koseri as a model organism for studying opportunistic infections

  • Creating experimental systems that mimic the immune environment of compromised hosts

The reported association between C. koseri pneumonia and underlying pulmonary adenocarcinoma suggests complex interactions between host pathology and bacterial opportunism that warrant further investigation .