Recombinant Shigella boydii serotype 4 ATP synthase subunit c (atpE)

What is ATP synthase subunit c (atpE) in Shigella boydii and what is its significance in bacterial physiology?

ATP synthase subunit c (atpE) is a critical component of the F0 sector of the F-type ATP synthase in Shigella boydii. This membrane-embedded protein forms an oligomeric ring structure that facilitates proton translocation across the bacterial membrane during ATP synthesis. The protein typically consists of 79-82 amino acids with a highly hydrophobic profile, containing predominantly alpha-helical secondary structures that span the membrane .

In bacterial physiology, atpE plays an essential role in energy metabolism by converting the proton gradient across the membrane into the mechanical energy needed for ATP synthesis. This function makes it indispensable for bacterial survival and growth, particularly under energy-limited conditions. Mutations in atpE can significantly impact bacterial bioenergetics and potentially affect virulence and antibiotic susceptibility.

What are the optimal conditions for expressing recombinant Shigella boydii serotype 4 atpE in E. coli expression systems?

Optimal expression of recombinant Shigella boydii atpE in E. coli requires careful consideration of several factors:

Expression System Components:

• Vector selection: pET vectors with T7 promoters are frequently used for membrane proteins like atpE due to their tight regulation and high expression potential

• E. coli strain: C41(DE3) or C43(DE3) strains are preferred for membrane proteins as they can accommodate the potentially toxic effects of membrane protein overexpression

• Fusion tags: N-terminal His-tags (as seen in the serotype 18 construct) facilitate purification while minimizing interference with membrane insertion

Optimized Expression Conditions:

• Temperature: Lower temperatures (16-25°C) often improve proper folding of membrane proteins

• Induction: Low concentrations of IPTG (0.1-0.5 mM) with longer expression times (16-24 hours)

• Media supplements: Addition of glucose (0.5-1%) to suppress basal expression and potential toxicity

• Aeration: Moderate aeration to balance growth and protein expression

The expression protocol should include careful monitoring of bacterial growth post-induction, as membrane protein overexpression can significantly impact cell viability.

What purification strategies are most effective for isolating recombinant atpE while maintaining its native conformation?

Purification of recombinant atpE presents significant challenges due to its hydrophobic nature and membrane association. The following multi-step approach is recommended:

• Membrane Isolation:

  • Cell disruption via French press or sonication in a buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl₂, and protease inhibitors

  • Differential centrifugation to separate membrane fractions

  • Solubilization of membranes using detergents

• Detergent Selection:

  • Mild detergents such as n-dodecyl-β-D-maltoside (DDM) or lauryl maltose neopentyl glycol (LMNG) at 1-2% concentration

  • Incubation at 4°C for 1-2 hours with gentle agitation

• Affinity Chromatography:

  • Immobilized metal affinity chromatography (IMAC) using the N-terminal His-tag

  • Wash buffer containing reduced detergent concentration (0.05-0.1%)

  • Elution with imidazole gradient (50-300 mM)

• Size Exclusion Chromatography:

  • Further purification by size exclusion chromatography

  • Buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03-0.05% detergent

• Protein Quality Assessment:

  • SDS-PAGE analysis for purity (>90% as standard)

  • Circular dichroism to verify alpha-helical secondary structure

  • Mass spectrometry for exact molecular weight determination

Maintaining protein stability during and after purification requires careful buffer optimization and proper storage conditions.

How can researchers troubleshoot low yields or poor solubility of recombinant atpE?

When encountering challenges with recombinant atpE expression and solubility, researchers should consider the following troubleshooting strategies:

For long-term storage, addition of 6% trehalose in Tris/PBS-based buffer at pH 8.0 has been effective for maintaining stability, with storage at -20°C/-80°C after aliquoting to avoid freeze-thaw cycles .

How does the structure and function of atpE contribute to Shigella pathogenesis and potential antibiotic targets?

While ATP synthase subunit c is primarily known for its role in energy metabolism, emerging research suggests connections between bacterial bioenergetics and pathogenesis:

Potential Pathogenesis Mechanisms:

• Energy production to support various virulence factor expression

• Maintenance of membrane potential which may influence secretion systems

• Adaptation to varying energy environments within the host

The structural features of atpE make it a potential antibiotic target. The oligomeric ring formed by multiple atpE subunits contains a conserved aspartate residue critical for proton translocation. This site has been targeted by antibiotics such as diarylquinolines, which bind to and inhibit mycobacterial ATP synthase. Similar approaches could potentially be developed for Shigella.

Research into the structural and functional relationships of atpE could inform novel antimicrobial development strategies specifically targeting Shigella infections, which would be especially valuable given the rising antibiotic resistance in Shigella species.

What experimental approaches are most effective for studying atpE interactions with other components of the ATP synthase complex?

Understanding atpE interactions within the ATP synthase complex requires sophisticated methodological approaches:

• Crosslinking Studies:

  • Chemical crosslinking combined with mass spectrometry to identify interaction points

  • Photo-activatable amino acid analogs for precise interaction mapping

  • Zero-length crosslinkers to identify direct protein-protein contacts

• Biophysical Methods:

  • Surface plasmon resonance (SPR) to determine binding kinetics

  • Microscale thermophoresis for quantifying molecular interactions

  • Isothermal titration calorimetry (ITC) for thermodynamic profiling

• Structural Biology Approaches:

  • Cryo-electron microscopy of the entire ATP synthase complex

  • Solid-state NMR for membrane-embedded interactions

  • X-ray crystallography of subcomplexes or the complete assembly

• Functional Assays:

  • ATP synthesis/hydrolysis assays to assess the impact of mutations

  • Proton translocation measurements using fluorescent probes

  • Reconstitution experiments in liposomes or nanodiscs

• Genetic Approaches:

  • Site-directed mutagenesis of interaction interfaces

  • Suppressor mutation analysis to identify compensatory changes

  • CRISPR-Cas9 genome editing to create variant proteins

These approaches provide complementary information that, when integrated, can yield comprehensive insights into how atpE contributes to ATP synthase function and potentially influences Shigella pathogenicity.

How can structural modifications of recombinant atpE affect its functional properties in experimental settings?

Strategic modifications to recombinant atpE can significantly impact its functional properties, enabling various experimental applications:

Key Structural Modifications:

• Site-Directed Mutagenesis:

  • Mutation of the conserved proton-binding aspartate residue to assess proton translocation mechanisms

  • Alteration of hydrophobic residues to examine membrane insertion and stability

  • Introduction of cysteine residues for site-specific labeling or crosslinking

• Fusion Proteins and Tags:

  • N-terminal His-tags for purification purposes have minimal impact on function when properly designed

  • Fluorescent protein fusions for localization studies may impact oligomerization

  • Split reporter systems to monitor protein-protein interactions in vivo

• Domain Swapping:

  • Exchange of segments between different bacterial species to assess species-specific functions

  • Creation of chimeric proteins to map functional domains

Functional Consequences of Modifications:

When designing modifications, researchers should consider the highly conserved nature of atpE and its critical role in bacterial survival. Validation experiments comparing modified atpE with wild-type protein are essential to ensure that observed effects are due to the specific modification rather than general structural disruption.

What are the most reliable methods for assessing the purity and integrity of recombinant atpE preparations?

Comprehensive quality assessment of recombinant atpE requires multiple complementary analytical techniques:

• SDS-PAGE Analysis:

  • Standard for initial purity assessment (>90% purity is typically recommended)

  • Both Coomassie and silver staining for different sensitivity levels

  • Western blotting with anti-His antibodies or specific anti-atpE antibodies

• Mass Spectrometry Approaches:

  • MALDI-TOF or ESI-MS for intact protein mass determination

  • Peptide mass fingerprinting after proteolytic digestion

  • Top-down proteomics for complete sequence verification

• Chromatographic Methods:

  • Size exclusion chromatography to assess oligomeric state and aggregation

  • Reverse-phase HPLC for purity assessment

  • Ion exchange chromatography to detect charge variants

• Spectroscopic Techniques:

  • Circular dichroism to verify alpha-helical secondary structure

  • Fluorescence spectroscopy to examine tertiary structure

  • FTIR for assessment of secondary structure components

• Functional Assays:

  • ATPase activity measurements when incorporated into ATP synthase complex

  • Proton translocation assays in reconstituted systems

  • Thermal shift assays to assess protein stability

The combination of multiple analytical techniques provides comprehensive characterization of recombinant atpE preparations, ensuring both structural integrity and functional competence.

What functional assays can be used to verify the activity of purified recombinant atpE?

Verifying the functional activity of purified recombinant atpE requires specialized assays that assess its ability to function within the ATP synthase complex:

• Reconstitution Experiments:

  • Incorporation of purified atpE into proteoliposomes

  • Co-reconstitution with other ATP synthase subunits to form functional complexes

  • Measurement of ATP synthesis driven by artificial proton gradients

• Proton Translocation Assays:

  • Fluorescent pH indicators to monitor proton movement

  • Potential-sensitive dyes to assess membrane potential changes

  • Radioisotope-based assays for precise quantification

• Binding Studies:

  • Surface plasmon resonance to measure interactions with other ATP synthase subunits

  • Isothermal titration calorimetry for thermodynamic characterization

  • Native mass spectrometry to observe complex formation

• Structural Integrity Assessment:

  • Oligomerization analysis using native PAGE or analytical ultracentrifugation

  • Electron microscopy to visualize ring formation

  • Atomic force microscopy for nanoscale structural analysis

• Inhibitor Binding Studies:

  • Measuring specific binding of known ATP synthase inhibitors

  • Competition assays with labeled ligands

  • Thermal shift assays in the presence of inhibitors

When interpreting results from these assays, it's important to consider that atpE functions as part of a complex, and its activity is interdependent with other ATP synthase components. Comparing results with those obtained using native ATP synthase provides important contextual information for interpreting recombinant protein function.

How can recombinant Shigella boydii atpE be utilized in vaccine development or diagnostic applications?

The potential applications of recombinant atpE extend beyond basic research into practical diagnostic and therapeutic developments:

Vaccine Development Applications:

• Recombinant atpE could serve as a carrier protein for antigenic epitopes

• While not a primary virulence factor, conserved membrane proteins like atpE may provide cross-protection against multiple serotypes

• Combination with other antigens in multicomponent vaccines could enhance protection

Diagnostic Applications:

• Development of serotype-specific antibodies against variable regions of atpE

• Creation of highly sensitive PCR primers targeting serotype-specific regions

• Potential component in multiplexed diagnostic assays for differentiation of Shigella species and serotypes

Technical Considerations:

• For vaccine applications, careful removal of all contaminating endotoxin is critical

• For diagnostics, high-purity preparations are essential to avoid cross-reactivity

• Expression systems need to be validated for diagnostic-grade or clinical-grade production

While not directly implicated in virulence like some other Shigella proteins (e.g., the StcE zinc metalloprotease found in atypical Shigella boydii 13) , the essential nature and conservation of atpE make it an interesting target for both diagnostic and therapeutic applications.

What are the emerging technologies that might enhance our understanding of atpE structure-function relationships?

Several cutting-edge technologies are poised to revolutionize our understanding of atpE biology:

These emerging technologies will provide unprecedented insights into the fundamental biology of atpE and potentially reveal new approaches for targeting ATP synthase in antimicrobial development.