Recombinant Bifidobacterium longum ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Bifidobacterium longum?

ATP synthase subunit c (atpE) in Bifidobacterium longum is a critical component of the F1F0-ATP synthase complex that catalyzes ATP production from ADP in the presence of a proton gradient. The atpE gene encodes for the c subunit, which forms the membrane-embedded oligomeric ring of the F0 portion of the ATP synthase. In B. longum, this enzyme plays a crucial role in energy metabolism, particularly under the anaerobic conditions of the human gut where this beneficial microorganism typically resides. As a lactic acid-producing bacterium, B. longum relies on efficient ATP production to maintain cellular functions in its natural environment .

The research methodology to study atpE typically involves genomic analysis to identify the gene sequence, followed by comparison with homologous proteins from related species. The complete genome sequences of several B. longum strains, including B. longum subsp. longum BBMN68 (available at NCBI database NC_014656.1), provide valuable resources for identifying and studying the atpE gene .

How does atpE function in the ATP synthase complex?

The c subunit (atpE) functions as part of the proton channel in the F0 portion of ATP synthase, facilitating proton translocation across the membrane. Multiple c subunits form a ring structure in the membrane, with each subunit containing an essential carboxyl group that participates in proton transport. The proton motive force drives the rotation of this c-ring, which is mechanically coupled to conformational changes in the F1 portion that catalyze ATP synthesis.

Research methodologies to investigate atpE function include:

• Site-directed mutagenesis of conserved residues to assess their role in proton translocation

• Reconstitution of the purified protein into liposomes to measure proton transport

• Analysis of ATP synthesis rates in relation to proton gradient magnitude

• Comparative genomics approaches to identify conserved functional domains across species

In B. longum specifically, the ATP synthase plays a crucial role in acid tolerance response (ATR), as evidenced by studies of ATPase activity under acidic conditions. For example, researchers have demonstrated that acid-adapted B. longum cells show increased ATPase activity compared to control cells, suggesting a role for ATP synthase in maintaining pH homeostasis .

What are the structural characteristics of atpE in B. longum?

The structural characteristics of atpE in B. longum can be determined through homology modeling, as direct crystallographic data specific to B. longum atpE is currently limited. The methodology for structural characterization typically involves:

• Sequence retrieval from databases such as NCBI

• Template identification using BLASTP against the Protein Data Bank

• Sequence alignment using tools like ClustalW to confirm sequence identity and similarity

• Homology modeling using software such as Modeller

• Energy minimization and refinement using molecular dynamics simulation

• Model evaluation using Ramachandran plots, ERRAT, and Verify_3D

Based on homology with related proteins, B. longum atpE likely consists of two transmembrane α-helices connected by a polar loop region. The first helix contains the conserved carboxyl group essential for proton binding. The c-ring formed by multiple atpE subunits creates a hydrophobic barrier that prevents proton leakage while facilitating controlled proton translocation coupled to ATP synthesis.

How is recombinant B. longum atpE typically produced in laboratory settings?

Recombinant B. longum atpE production typically employs the following methodological approach:

• Gene amplification: The atpE gene is amplified from B. longum genomic DNA using PCR with specific primers designed based on the available genome sequence .

• Expression vector construction: The amplified gene is cloned into an appropriate expression vector, typically containing:

  • A strong, inducible promoter (e.g., T7 or tac)

  • A fusion tag to facilitate purification (His6, GST, or MBP)

  • Appropriate selection markers

• Expression optimization: Several parameters require optimization:

  • Host strain selection (E. coli BL21(DE3), C41(DE3), or C43(DE3) for membrane proteins)

  • Induction conditions (temperature, inducer concentration, duration)

  • Media composition (including potential supplementation with specific ions)

• Membrane protein extraction: As atpE is a membrane protein, specialized extraction protocols using detergents are necessary, with typical steps including:

  • Cell lysis by sonication or French press

  • Membrane fraction isolation by ultracentrifugation

  • Selective solubilization using mild detergents (DDM, CHAPS, or LDAO)

• Purification strategy: A multi-step purification approach including:

  • Affinity chromatography based on the fusion tag

  • Size-exclusion chromatography

  • Ion-exchange chromatography if needed

Yield verification typically involves SDS-PAGE, Western blotting, and activity assays to confirm both quantity and functionality of the purified protein.

What are the optimal expression systems for recombinant B. longum atpE?

Optimizing expression systems for recombinant B. longum atpE requires careful consideration of several methodological aspects:

• Prokaryotic vs. Eukaryotic Expression:

  • E. coli systems remain the most widely used for initial attempts due to rapid growth and high protein yields

  • Specialized strains like C41(DE3) and C43(DE3) designed for membrane protein expression show improved results for ATP synthase components

  • Lactococcus lactis has emerged as an alternative host for expressing proteins from lactic acid bacteria, potentially providing a more native-like membrane environment

• Vector Design Considerations:

  • Codon optimization based on the host's preferential codon usage

  • Inclusion of fusion partners that enhance solubility (MBP, SUMO)

  • Incorporation of cleavable tags that can be removed post-purification

  • Tight regulation of expression to prevent toxicity

• Expression Conditions:

  • Lower temperatures (16-25°C) to slow protein production and enhance proper folding

  • Reduced inducer concentrations

  • Extended expression periods (overnight to 48 hours)

  • Supplementation with specific lipids that might stabilize the membrane protein

• Cell-Free Expression Systems:

  • Emerging approach for difficult membrane proteins

  • Direct synthesis into artificial liposomes or nanodiscs

  • Allows immediate incorporation into a lipid environment

  • Eliminates toxicity issues associated with overexpression in living cells

Each approach requires systematic optimization, with expression levels monitored via Western blotting and activity assays to confirm functional protein production.

How does acid stress affect atpE expression and function in B. longum?

Research on acid stress in Bifidobacterium longum provides important insights into atpE expression and function. The methodological approach to investigating this question involves:

• Acid Adaptation Protocols:
  Studies have demonstrated that B. longum can develop acid tolerance response (ATR) when pre-exposed to sublethal acidic conditions. For example, acid adaptation at pH 4.5 for 2 hours significantly increased the survival rate of B. longum subsp. longum BBMN68 at lethal pH 3.5 .

• Gene Expression Analysis:
  RNA-Seq analysis of acid-adapted B. longum cells revealed comprehensive gene expression changes. In BBMN68, the expression of 538 genes changed by more than 2-fold after acid adaptation, with 293 genes upregulated .

• ATPase Activity Measurement:
  Analysis of ATPase activity in acid-adapted cells compared to control cells provides direct evidence of functional changes. The methodology typically involves:

  • Cell collection and washing with MgCl2

  • Measurement of ATPase activity using standardized enzyme assays

  • Correlation of activity with gene expression data

• Correlation Analysis:
  Verification of RNA-Seq data with RT-PCR shows strong correlation (R² = 0.96 in one study), confirming the reliability of expression profiles .

What structural modifications can enhance the stability of recombinant atpE?

Enhancing the stability of recombinant atpE involves several structural modification approaches:

• Site-Directed Mutagenesis Strategies:

  • Identification of destabilizing residues through computational prediction tools

  • Introduction of disulfide bridges at strategic positions

  • Replacement of surface-exposed hydrophobic residues with polar ones

  • Proline substitutions in loop regions to reduce flexibility

• Fusion Partner Selection:

  • N-terminal or C-terminal fusion with thermostable proteins

  • Incorporation of rigid linkers between fusion partners

  • Selection of fusion partners based on compatible folding pathways

• Membrane-Mimetic Environments:

  • Identification of optimal detergent types and concentrations

  • Reconstitution into nanodiscs with defined lipid composition

  • Incorporation into amphipols or styrene-maleic acid lipid particles (SMALPs)

• Computational Design Approaches:

  • Molecular dynamics simulations to identify regions of high mobility

  • Energy minimization analysis similar to that used for AtpE modeling

  • Rosetta-based design of stabilizing mutations

The effectiveness of these modifications can be evaluated through thermal shift assays, proteolytic resistance tests, and long-term activity measurements to quantify improvements in stability while maintaining functional properties.

How can atpE be targeted for potential antimicrobial development?

The methodological approach to targeting atpE for antimicrobial development involves:

• Target Validation:

  • Confirmation of essentiality through conditional gene knockdown systems

  • Demonstration of growth inhibition when atpE function is compromised

  • Evolutionary conservation analysis to assess selective targeting potential

• Structure-Based Drug Design:

  • Development of homology models as demonstrated for AtpE from Mycobacterium tuberculosis

  • Energy minimization and refinement using molecular dynamics simulation

  • Virtual screening against large compound libraries using RASPD and PyRx tools

• Compound Screening Workflow:

  • Initial high-throughput virtual screening for binding energy assessment

  • Filtering by Lipinski's rule of five to ensure drug-like properties

  • Molecular docking analysis using tools like AutoDock4.2

  • Analysis of protein-ligand complexes via Pymol and Ligplot+

• Lead Optimization:

  • Structure-activity relationship studies based on initial hits

  • Modification of promising compounds to enhance binding affinity

  • Assessment of specificity for bacterial versus human ATP synthase

Studies on AtpE inhibitors for Mycobacterium tuberculosis identified compounds with binding energies ranging from -8.69 to -8.44 kcal/mol, demonstrating stronger binding than ATP itself . Similar approaches could be applied to B. longum atpE, though with careful consideration of the potential impact on beneficial gut microbiota.

What are the challenges in crystallizing recombinant B. longum atpE for structural studies?

Crystallization of membrane proteins like B. longum atpE presents several methodological challenges:

• Protein Production Challenges:

  • Low expression levels typical of membrane proteins

  • Potential toxicity to host cells during overexpression

  • Difficulty maintaining native conformation during extraction

  • Limited stability in detergent solutions

• Purification Obstacles:

  • Selection of appropriate detergents that maintain protein stability

  • Prevention of protein aggregation during concentration steps

  • Removal of lipid contaminants that might interfere with crystal contacts

  • Achieving sufficient purity (>95%) required for crystallization

• Crystallization Technique Selection:

  • Vapor diffusion methods (hanging drop, sitting drop)

  • Lipidic cubic phase methods for membrane proteins

  • Bicelle-based crystallization

  • Microseeding approaches to overcome nucleation barriers

• Crystal Optimization Strategies:

  • Screening of various precipitants, buffers, and additives

  • Temperature and pH variation

  • Addition of lipids or detergents that stabilize crystal contacts

  • Construction of fusion proteins with crystallization chaperones

• Alternative Structural Approaches:

  • Cryo-electron microscopy for the entire ATP synthase complex

  • NMR studies of isolated domains or labeled proteins

  • Small-angle X-ray scattering for low-resolution envelope determination

Each challenge requires systematic optimization, with techniques analogous to those used for structure determination of ATP synthase components from other organisms.

What are the best protocols for purifying recombinant B. longum atpE while maintaining its native conformation?

Purification of recombinant B. longum atpE with preserved native conformation involves a carefully designed methodological workflow:

• Membrane Fraction Isolation:

  • Cell disruption under mild conditions (low pressure homogenization)

  • Differential centrifugation to collect membrane fractions

  • Washing steps to remove peripheral membrane proteins

• Detergent Selection and Optimization:

  • Screening of multiple detergents (DDM, LMNG, CHAPS)

  • Determination of critical micelle concentration for each detergent

  • Gradual solubilization to minimize denaturation

  • Assessment of protein activity retention in each detergent

• Chromatographic Purification Strategy:

  • Initial capture via affinity chromatography (IMAC for His-tagged constructs)

  • Intermediate purification via ion exchange chromatography

  • Polishing step using size exclusion chromatography

  • All buffers containing stabilizing detergent at appropriate concentrations

• Quality Control Assessments:

  • Circular dichroism spectroscopy to confirm secondary structure

  • Thermal shift assays to evaluate stability

  • Activity assays specific to proton translocation function

  • Blue native PAGE to assess oligomeric state

• Reconstitution Methods:

  • Controlled detergent removal via dialysis or adsorption

  • Incorporation into liposomes of defined lipid composition

  • Proton pumping activity verification in the reconstituted system

This comprehensive approach ensures both purity and functionality of the isolated protein, critical for subsequent structural and functional studies.

How can site-directed mutagenesis be used to study the functional domains of B. longum atpE?

Site-directed mutagenesis provides a powerful methodological approach to dissect the structure-function relationships in B. longum atpE:

• Target Residue Identification Strategy:

  • Sequence alignment with homologous proteins from well-studied organisms

  • Identification of conserved residues across species

  • Structural modeling to predict functional domains

  • Special focus on the essential carboxyl residue involved in proton binding

• Mutagenesis Experimental Design:

  • Alanine scanning mutagenesis of conserved residues

  • Conservative versus non-conservative substitutions

  • Creation of chimeric proteins with sections from related species

  • Introduction of reporter groups at specific positions

• Technical Mutagenesis Approaches:

  • QuikChange PCR-based mutagenesis

  • Gibson Assembly for multiple simultaneous mutations

  • Golden Gate Assembly for systematic domain swapping

  • Verification by sequencing before protein expression

• Functional Assessment Methodology:

  • Expression and purification using protocols optimized for wild-type protein

  • ATP synthesis activity measurements in reconstituted systems

  • Proton translocation assays using pH-sensitive fluorophores

  • Structural integrity verification via circular dichroism

• Data Interpretation Framework:

  • Classification of mutations as neutral, partially disruptive, or completely disruptive

  • Mapping of functional importance onto structural models

  • Correlation analysis between conservation level and functional impact

  • Development of a comprehensive functional map of the protein

This systematic approach has been successfully employed for ATP synthase components in other organisms and can be adapted specifically for B. longum atpE.

What assays are most reliable for measuring ATP synthase activity in recombinant systems?

Several methodological approaches provide reliable measurements of ATP synthase activity in recombinant systems:

• ATP Synthesis Assays:

  • Luciferase-based luminescence detection of ATP production

  • Reconstitution of purified enzyme into liposomes

  • Generation of artificial proton gradient using pH jump or valinomycin/K+ systems

  • Continuous monitoring of ATP production rates

  • Controls including ionophores to collapse proton gradients

• ATP Hydrolysis Assays:

  • Spectrophotometric coupled enzyme assays (with pyruvate kinase and lactate dehydrogenase)

  • Colorimetric detection of released phosphate

  • Measurement of ATPase activity as demonstrated for B. longum under acid stress conditions

  • Inhibitor studies to confirm specificity (oligomycin, DCCD)

• Proton Translocation Measurements:

  • Fluorescent pH indicators (ACMA, pyranine) in proteoliposomes

  • Measurement of fluorescence quenching upon energization

  • Stopped-flow rapid kinetics for initial rate determination

  • Confirmation of coupling between proton movement and catalysis

• Structural Integrity Assessments:

  • Blue native PAGE to verify intact complex assembly

  • Electron microscopy to visualize reconstituted complexes

  • FRET-based approaches to monitor conformational changes during catalysis

• In Vivo Complementation:

  • Expression in ATP synthase-deficient strains

  • Growth rate comparison under conditions requiring oxidative phosphorylation

  • Membrane potential measurements in whole cells

Each assay provides complementary information, and combining multiple approaches yields the most comprehensive assessment of enzymatic function.

How can discrepancies in atpE function between in vitro and in vivo studies be reconciled?

Reconciling discrepancies between in vitro and in vivo studies of atpE function requires a multi-faceted methodological approach:

• Systematic Comparison Framework:

  • Parallel measurements under standardized conditions

  • Identification of specific parameters showing discrepancies

  • Development of intermediate "semi-in vivo" systems (spheroplasts, right-side-out vesicles)

• Environmental Factor Analysis:

  • Reconstitution of in vitro systems with native lipid compositions

  • Incorporation of molecular crowding agents to mimic cytoplasmic conditions

  • Adjustment of pH, ion concentrations, and metabolite levels to physiological values

  • Temperature optimization to match growth conditions

• Interaction Network Mapping:

  • Identification of protein-protein interactions present in vivo but absent in vitro

  • Copurification strategies to maintain protein complexes

  • Addition of stabilizing factors identified from proteomic studies

  • Assessment of post-translational modifications present in vivo

• Methodological Refinement:

  • Development of whole-cell assays with higher resolution

  • Improvement of protein extraction methods to preserve native states

  • Application of techniques like RNA-Seq to correlate functional changes with expression patterns

  • Use of cryo-electron tomography to visualize complexes in their native membrane environment

• Mathematical Modeling Approaches:

  • Development of models integrating both in vitro kinetic parameters and in vivo constraints

  • Sensitivity analysis to identify key parameters causing discrepancies

  • Iterative model refinement based on experimental validation

This comprehensive approach has successfully reconciled similar discrepancies for other membrane proteins and can be applied specifically to the study of B. longum atpE.

What statistical approaches are most appropriate for analyzing comparative studies of wild-type versus recombinant atpE?

Analyzing comparative studies of wild-type versus recombinant atpE requires rigorous statistical methodologies:

• Experimental Design Considerations:

  • Power analysis to determine appropriate sample sizes

  • Blocking and randomization to minimize systematic errors

  • Inclusion of multiple technical and biological replicates

  • Consideration of paired designs when appropriate

• Data Quality Assessment:

  • Normality testing of distributions (Shapiro-Wilk, Kolmogorov-Smirnov)

  • Homogeneity of variance evaluation (Levene's test, Bartlett's test)

  • Identification and handling of outliers (Grubbs' test, Dixon's Q test)

  • Assessment of technical variability through coefficient of variation

• Comparative Statistical Methods:

  • Parametric approaches:

    • Student's t-test for single parameter comparisons

    • ANOVA with post-hoc tests for multiple comparisons

    • ANCOVA when controlling for covariates

  • Non-parametric alternatives:

    • Mann-Whitney U test or Wilcoxon signed-rank test

    • Kruskal-Wallis with Dunn's post-test

• Multivariate Analysis Techniques:

  • Principal Component Analysis to identify patterns in multidimensional data

  • Hierarchical clustering to group similar variants

  • Partial Least Squares Discriminant Analysis for classification

• Regression Modeling:

  • Linear models for continuous outcomes

  • Generalized linear models for non-normal distributions

  • Mixed effects models to account for repeated measures

• Validation Approaches:

  • Cross-validation techniques to assess model robustness

  • Bootstrapping for confidence interval estimation

  • Permutation tests for significance assessment

For RNA-Seq data analysis specifically, the DEGseq approach has been successfully employed in B. longum studies to normalize expression levels using RPKM values and identify differentially expressed genes with statistical confidence (p<0.001) .