Recombinant Aquifex aeolicus Flagellar biosynthetic protein FliQ (fliQ)

What is Aquifex aeolicus and why is its FliQ protein of interest?

Aquifex aeolicus is a chemolithoautotrophic, Gram-negative, motile, hyperthermophilic bacterium that belongs to the Aquificota phylum, one of the earliest diverging bacterial lineages . This rod-shaped organism (2.0-6.0μm in length, 0.4-0.5μm in diameter) thrives in extreme environments with temperatures between 85°C and 95°C, typically near underwater volcanoes or hot springs . Its flagellar system, including the FliQ protein, has evolved to function under these extreme conditions, making it an excellent model for studying protein stability and function at high temperatures. The FliQ protein specifically forms part of the FliPQR complex that serves as a crucial channel for the export of flagellar proteins and as a template for flagellar rod assembly .

What are the structural characteristics of recombinant A. aeolicus FliQ protein?

The recombinant A. aeolicus FliQ protein consists of 89 amino acids with the sequence: MEQDLIVSLGQRALEMTLLLALPVLLSTFVVGLVVSIFQAATQIQEMTLSYIPKVITAFLVIFLLGGWMMRKLVDFAVEIFANIPVWIR . The protein is typically produced with an N-terminal His-tag to facilitate purification . As part of the FliPQR complex, FliQ contributes to the formation of a channel structure that participates in flagellar protein export . The N-terminal regions of the complex components (including FliP and FliR) form a periplasmic gate structure, with a β-cap created by the N-terminal β-strands creating a tight seal in the closed conformation . The relatively small size of FliQ (89 amino acids) suggests it likely serves a specialized structural or regulatory role within the flagellar assembly system.

How is recombinant A. aeolicus FliQ typically expressed and purified?

Recombinant A. aeolicus FliQ is commonly expressed in E. coli expression systems due to their efficiency and scalability . The gene encoding the full-length FliQ protein (amino acids a1-89) is cloned into an appropriate expression vector with an N-terminal His-tag for purification purposes . Following expression, the protein is typically purified using affinity chromatography, where the His-tagged protein binds to nickel or cobalt resin, allowing contaminants to be washed away before elution with imidazole-containing buffer. The purified protein is then dialyzed to remove imidazole and concentrated. The final product is often lyophilized for long-term storage and stability . For researchers studying the FliPQR complex, co-expression systems may be employed to produce the entire complex, similar to approaches used for other multi-subunit A. aeolicus proteins such as the heterodimeric LeuRS .

What are the recommended storage conditions for recombinant FliQ?

Based on established protocols for recombinant A. aeolicus FliQ, the lyophilized protein should be stored at -20°C to -80°C upon receipt . After reconstitution, working aliquots can be stored at 4°C for up to one week . For long-term storage of reconstituted protein, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being standard) and store aliquots at -20°C to -80°C . Repeated freeze-thaw cycles should be avoided to prevent protein degradation and loss of activity . When reconstituting the lyophilized protein, it is advised to use deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL . The standard storage buffer for reconstituted protein is Tris/PBS-based buffer with 6% trehalose at pH 8.0, which helps maintain protein stability .

How does FliQ function within the FliPQR complex during flagellar assembly?

The FliPQR complex, which includes FliQ, constitutes a critical channel for the export of bacterial flagellar proteins involved in axial structure assembly and serves as a template for flagellar rod assembly . Recent cryoEM structural studies at 3.0 Å resolution have revealed that the complex features a periplasmic gate formed by the N-terminal α-helices of FliP and FliR, which remains closed until FliE assembles onto FliP and FliR . The N-terminal β-strands of FliP and FliR create a β-cap that forms a tight seal in the closed gate configuration . When FliE interacts with FliP and FliR, it induces a conformational change causing their N-terminal α-helices to move upward and outward . This movement initiates the sequential opening of the periplasmic gate as the N-terminal β-strands of FliP and FliR begin to separate, creating a docking site for FliE and enabling rod assembly to proceed . FliQ's precise role in this mechanism likely involves stabilizing the complex and facilitating the conformational changes necessary for gate opening.

How can researchers investigate temperature-dependent structural changes in A. aeolicus flagellar proteins?

To investigate temperature-dependent structural changes in A. aeolicus flagellar proteins like FliQ, researchers can apply methodologies similar to those used in studies of other A. aeolicus proteins. For example, with the FlgM protein, circular dichroism (CD) experiments revealed that at 20°C, the protein exhibits alpha-helical character, but the percentage of alpha-helical content decreases with increased temperature, indicating a transition to a less folded conformation at higher temperatures . The same study employed chemical denaturation experiments to demonstrate cooperativity consistent with a globular nature at lower temperatures . Additionally, researchers used the fluorescent probe FlAsH to show specific conformational states of protein helices at different temperatures . For FliQ studies, similar approaches could be implemented to examine how the protein's structure changes between room temperature and A. aeolicus' physiological temperature of 85°C . These methods could reveal whether FliQ, like FlgM, adopts a more compact structure at lower temperatures that becomes more extended at higher temperatures, which would have important implications for understanding how the flagellar export system functions in this thermophilic organism.

What experimental approaches can be used to study the interaction between FliQ and other components of the flagellar export apparatus?

Several experimental approaches can be employed to study the interactions between FliQ and other components of the flagellar export apparatus:

• Co-immunoprecipitation (Co-IP): Using antibodies against tagged versions of FliQ to pull down protein complexes, followed by western blotting or mass spectrometry to identify interaction partners.

• Bacterial Two-Hybrid System: Fusing FliQ and potential interaction partners to complementary fragments of a reporter protein (like adenylate cyclase) to detect interactions based on reporter activity.

• Crosslinking studies: Using chemical crosslinkers to stabilize transient protein interactions before analysis by western blotting or mass spectrometry.

• Surface Plasmon Resonance (SPR): Measuring real-time binding kinetics between immobilized FliQ and other components in solution.

• Structural studies: Employing cryoEM methods similar to those used for the FliPQR complex to visualize FliQ within larger assemblies at different functional states.

• Protein fusion experiments: Creating hybrid proteins between A. aeolicus and E. coli components, similar to approaches used with LeuRS , to determine which regions are critical for specific interactions.

• Site-directed mutagenesis: Systematically altering conserved residues in FliQ to identify amino acids critical for complex formation and function.

These approaches, often used in combination, can provide comprehensive insights into how FliQ interacts with FliP, FliR, and other components in the context of flagellar assembly and protein export.

What expression systems are most effective for producing functional recombinant A. aeolicus FliQ?

The most effective expression systems for producing functional recombinant A. aeolicus FliQ must accommodate the challenges associated with expressing thermophilic proteins in mesophilic hosts. Based on established protocols, E. coli expression systems have proven effective for FliQ production . Key considerations include:

• Expression strain selection: BL21(DE3) or derivatives are commonly used due to their reduced protease activity and capability for high-level expression.

• Codon optimization: Adapting the A. aeolicus FliQ gene sequence to E. coli codon usage can significantly improve expression levels.

• Fusion tags: The N-terminal His-tag is standard for purification purposes , but other fusion partners like MBP (maltose-binding protein) or SUMO might improve solubility.

• Induction conditions: Lower induction temperatures (16-25°C) often improve solubility of thermophilic proteins in E. coli, despite seeming counterintuitive.

• Co-expression strategies: For studies of the FliPQR complex, co-expression of all three components might improve proper folding and complex formation, similar to approaches used for other A. aeolicus protein complexes .

• Cell-free expression systems: These can be advantageous for toxic or difficult-to-express proteins and allow immediate incorporation into liposomes for functional studies.

The choice between these systems depends on the specific research goals, whether studying FliQ in isolation or as part of the FliPQR complex.

How should researchers account for the thermophilic origin of A. aeolicus when designing experiments?

When designing experiments involving A. aeolicus FliQ, researchers must account for its thermophilic origin with several important considerations:

• Temperature selection: While A. aeolicus grows optimally between 85-95°C , research shows that its proteins may exhibit different conformational states at different temperatures . Experiments should be conducted at both the physiological temperature (85°C) and standard laboratory temperatures to understand temperature-dependent behavior.

• Buffer stability: Buffers with temperature-dependent pKa values (like Tris) may change pH significantly at high temperatures. Phosphate buffers or other temperature-stable buffers should be considered for high-temperature experiments.

• Protein stability assessment: Prior to functional assays, thermal stability studies (using techniques like differential scanning calorimetry) should be performed to determine the temperature range where the recombinant protein maintains its native structure.

• Comparative controls: Including mesophilic homologs as controls can help distinguish general protein properties from thermophile-specific adaptations.

• Equipment limitations: Standard laboratory equipment may not function properly at extreme temperatures. Specialized high-temperature incubators and reaction vessels may be required.

• Experimental timeline adjustments: Reaction rates typically increase with temperature, so kinetic studies at physiological temperatures for A. aeolicus may require shorter timepoints than would be used for mesophilic proteins.

These considerations ensure that experimental conditions appropriately reflect the native environment of A. aeolicus FliQ while accommodating practical laboratory constraints.

How can researchers overcome solubility issues when working with recombinant FliQ?

Membrane-associated proteins like FliQ often present solubility challenges during recombinant expression and purification. Researchers can employ several strategies to overcome these issues:

• Optimization of expression conditions: Reducing expression temperature (16-25°C), lowering inducer concentration, and using specialized E. coli strains designed for membrane protein expression can improve solubility.

• Fusion partners: Employing solubility-enhancing fusion partners such as MBP, SUMO, or TrxA can significantly improve soluble yields. These can be removed later with specific proteases if necessary.

• Detergent screening: Systematic testing of different detergents (non-ionic, zwitterionic, and mild ionic) can identify optimal conditions for extracting and maintaining FliQ in solution. Common options include DDM, LDAO, or Triton X-100.

• Lipid nanodisc incorporation: Reconstituting purified FliQ into lipid nanodiscs can provide a native-like membrane environment while maintaining solubility in aqueous solutions.

• Co-expression with partners: Expressing FliQ together with its natural interaction partners (FliP and FliR) may improve proper folding and solubility, as the proteins may stabilize each other in their native complex .

• Refolding protocols: If inclusion bodies form, carefully optimized refolding protocols using gradual removal of denaturants can sometimes recover properly folded protein.

• Buffer optimization: Screening different buffer compositions, salt concentrations, pH values, and additives (glycerol, trehalose, arginine) can identify conditions that enhance solubility.

These approaches can be tested systematically or in combination to determine the optimal conditions for producing soluble, functional recombinant FliQ.

What are the most effective methods for reconstituting lyophilized FliQ protein?

For optimal reconstitution of lyophilized A. aeolicus FliQ protein, researchers should follow these methodological steps:

• Pre-reconstitution preparation: Briefly centrifuge the vial containing lyophilized protein to ensure all material is at the bottom of the container .

• Selection of reconstitution buffer: Use deionized sterile water for initial reconstitution to achieve a concentration of 0.1-1.0 mg/mL . For applications requiring specific buffer conditions, consider reconstituting in a concentrated buffer that can be diluted to the desired final concentration.

• Reconstitution process: Add the reconstitution solution slowly to the lyophilized powder, allowing it to wet completely before gentle mixing. Avoid vigorous vortexing which can cause protein denaturation or aggregation.

• Concentration determination: Measure protein concentration using spectrophotometric methods (A280) or colorimetric assays (Bradford or BCA) after reconstitution to confirm recovery.

• Glycerol addition: For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being standard) . This helps prevent freeze-thaw damage during storage.

• Aliquoting: Divide the reconstituted protein into small single-use aliquots to avoid repeated freeze-thaw cycles .

• Storage conditions: Store working aliquots at 4°C for up to one week or at -20°C/-80°C for long-term storage .

• Quality control: Before experimental use, verify protein integrity by SDS-PAGE and/or functional assays appropriate to your research application.

Following these protocols maximizes the recovery of functionally active FliQ protein from lyophilized preparations.

How does A. aeolicus FliQ compare to FliQ proteins from mesophilic bacteria?

A comparative analysis of A. aeolicus FliQ with its mesophilic counterparts reveals several important adaptations that likely contribute to its function at extreme temperatures:

• Amino acid composition: A. aeolicus FliQ likely contains a higher proportion of charged residues (particularly glutamic acid and arginine) and fewer thermolabile residues (like asparagine and glutamine) compared to mesophilic homologs, a common adaptation in thermophilic proteins.

• Structural stability: Like other A. aeolicus proteins, FliQ probably exhibits increased intramolecular interactions (ionic bonds, hydrogen bonds, and hydrophobic interactions) that maintain structural integrity at high temperatures.

• Conformational flexibility: Studies on other A. aeolicus proteins like FlgM suggest that thermophilic proteins may adopt different conformational states at different temperatures, with more compact structures at lower temperatures and more extended conformations at physiological temperatures (85°C) . FliQ may exhibit similar temperature-dependent conformational dynamics.

• Complex formation: Within the FliPQR complex, the interactions between A. aeolicus FliQ and its partner proteins (FliP and FliR) are likely stronger than in mesophilic systems to maintain complex integrity at high temperatures .

• Evolutionary conservation: The core functional regions of FliQ are likely conserved across species, while the thermostabilizing features represent adaptations specific to thermophilic organisms.

This comparative perspective provides valuable insights into both the fundamental mechanisms of protein thermostability and the evolutionary adaptations that allow life to thrive in extreme environments.

What evolutionary insights can be gained from studying FliQ in ancient thermophilic bacteria?

Studying FliQ in Aquifex aeolicus offers significant evolutionary insights due to this organism's position as one of the earliest diverging bacterial lineages . Key evolutionary perspectives include:

• Ancestral traits: A. aeolicus is thought to represent one of the oldest bacterial lineages, related to filamentous bacteria first observed at the turn of the last century . Its flagellar system, including FliQ, may therefore preserve characteristics of ancient flagellar export systems.

• Thermophilic origins hypothesis: The thermostability of A. aeolicus proteins supports theories about the thermophilic origin of life, suggesting that early cellular life evolved in high-temperature environments similar to deep-sea hydrothermal vents.

• Protein interaction network evolution: Comparing the FliPQR complex structure and function across phylogenetically diverse bacteria can reveal how protein-protein interactions evolve while maintaining critical cellular functions.

• Minimal functional requirements: A. aeolicus, with its relatively small genome adapted to extreme conditions, likely retains only essential components of the flagellar system. Studying its FliQ and related proteins helps identify the core elements required for flagellar function.

• Convergent vs. divergent evolution: Comparing thermostability mechanisms in A. aeolicus FliQ with those in unrelated thermophiles (like thermophilic archaea) can distinguish between ancestral features and convergently evolved adaptations to high temperatures.

• Molecular clock applications: The degree of sequence conservation between A. aeolicus FliQ and homologs in other bacteria can contribute to molecular clock analyses that help date evolutionary divergences in the bacterial domain.

These evolutionary insights extend beyond FliQ itself to broader questions about bacterial evolution and adaptation to extreme environments.

Table 1: Key Properties of Recombinant A. aeolicus FliQ Protein

Table 2: Comparative Analysis of A. aeolicus FliQ with Related Proteins