Recombinant Aquifex aeolicus Flagellar protein FliL (fliL)

What is the biological role of FliL in Aquifex aeolicus?

FliL is a flagellar protein found in the hyperthermophilic bacterium Aquifex aeolicus that appears to play a regulatory role in flagellar motor function. While initially thought to be essential for motility in some bacterial species, comprehensive analysis suggests FliL functions as a negative modulator of motor activity rather than being strictly required for flagellar rotation. The protein contains a transmembrane domain that is critical for its functional properties and interaction with other flagellar components . A. aeolicus uses its flagella to achieve swimming speeds of approximately 90 μm/s at its optimal growth temperature of 85°C .

How does A. aeolicus FliL compare with homologs from other bacterial species?

Sequence analysis of FliL proteins across different bacterial species reveals that while there is significant structural conservation in the transmembrane regions, there are notable variations in phenotypic expressions when FliL is mutated. Unlike some mesophilic bacteria where FliL disruption may completely abolish motility, partial deletion of FliL in thermophiles like A. aeolicus can sometimes enhance motility, particularly under high-viscosity conditions such as elevated agar concentrations (0.5-1%) . This suggests that the protein's role may have evolved differently in extremophiles compared to their mesophilic counterparts.

What are the structural characteristics of A. aeolicus FliL?

The FliL protein from A. aeolicus contains a well-defined transmembrane domain that anchors it to the cell membrane. Computational prediction tools such as TMHMM identify specific transmembrane regions that are highly conserved . Similar to other A. aeolicus proteins, FliL likely exhibits temperature-dependent conformational changes, potentially adopting a more ordered structure at lower temperatures (around 20°C) and transitioning to a more extended, functional conformation at the organism's physiological temperature range (85-95°C) .

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

For successful expression of recombinant A. aeolicus FliL, E. coli-based heterologous expression systems have proven effective, particularly when employing temperature-inducible promoters. When expressing thermophilic proteins in mesophilic hosts, researchers should consider using E. coli strains optimized for expression of proteins with rare codons (such as Rosetta or CodonPlus strains). Expression trials suggest maintaining growth temperatures at 30°C during induction phase to balance protein yield and proper folding, followed by heat treatment steps (70-80°C) for purification advantage, as most E. coli proteins will denature while A. aeolicus proteins remain stable .

What purification strategies yield the highest purity and activity for recombinant A. aeolicus FliL?

A multi-step purification approach is recommended for recombinant A. aeolicus FliL:

• Heat treatment (75-80°C for 20 minutes) to exploit thermostability

• Immobilized metal affinity chromatography (IMAC) using His-tagged constructs

• Size exclusion chromatography for final polishing

This protocol typically yields protein with >95% purity suitable for functional and structural studies. When designing constructs, consider that full-length FliL with its transmembrane domain may require detergent solubilization, while truncated versions (ΔfliL1) lacking the transmembrane region can be expressed as soluble proteins but may have altered functional properties .

How can researchers effectively assess the functional properties of A. aeolicus FliL?

To comprehensively evaluate FliL function, researchers should employ a combination of approaches:

• Complementation studies: Express A. aeolicus FliL in FliL-deficient strains of model organisms (e.g., E. coli) and assess motility restoration on soft agar plates (0.3-0.5% agar) .

• Chimeric protein analysis: Create fusion proteins combining the transmembrane domain of E. coli FliL with the cytoplasmic domains of A. aeolicus FliL to identify functionally critical regions.

• Site-directed mutagenesis: Target conserved residues, particularly in the transmembrane region and cytoplasmic domain, as point mutations in these regions have been shown to significantly alter flagellar function and motility .

• Temperature-dependent motility assays: Evaluate swimming behavior at different temperatures (20-85°C) using specialized high-temperature microscopy setups to understand thermoadaptation of flagellar function .

What techniques can be used to study protein-protein interactions involving A. aeolicus FliL?

Several methods have proven effective for studying FliL interactions with other flagellar components:

• Bacterial two-hybrid assays: Adapted for high-temperature interactions by using thermostable reporter systems.

• Co-immunoprecipitation: Using antibodies against FliL or potential binding partners, ideally performed under conditions that preserve native protein states.

• Surface plasmon resonance (SPR): For quantitative measurement of binding kinetics with purified interaction partners.

• Crosslinking studies: Chemical crosslinking followed by mass spectrometry to identify interacting residues and proteins in vivo.

These approaches have revealed that FliL interacts with both the stator complex (MotA/MotB) and components of the rotor, suggesting it may function at the interface between these structures .

What structural determination methods are most suitable for A. aeolicus FliL?

Recent advances in structural biology techniques offer several approaches for characterizing A. aeolicus FliL:

• X-ray crystallography: Most effective for soluble domains of FliL, requiring optimization of crystallization conditions for thermophilic proteins.

• Cryo-electron microscopy (cryo-EM): Particularly useful for visualizing FliL in the context of the entire flagellar complex. Recent advancements have achieved near-atomic resolution (1.42 Å) for other A. aeolicus proteins, suggesting this approach could yield high-resolution structures of flagellar components .

• NMR spectroscopy: Suitable for studying the dynamics of specific domains and their temperature-dependent conformational changes.

• Molecular dynamics simulations: Computational approaches to predict structure and conformational changes at different temperatures, validated against experimental data.

The feasibility of these methods can be assessed based on recent successes with other A. aeolicus proteins. For example, the lumazine synthase from A. aeolicus was recently characterized at 1.42 Å resolution using cryo-EM, showing the potential of this technique for high-resolution structural studies of proteins from this organism .

How can researchers investigate temperature-dependent conformational changes in A. aeolicus FliL?

To study thermally induced structural transitions in FliL, researchers should consider:

• Circular dichroism (CD) spectroscopy: To monitor secondary structure changes across a temperature range (20-95°C), similar to studies on A. aeolicus FlgM that revealed decreased α-helical content at elevated temperatures .

• Differential scanning calorimetry (DSC): To determine thermal transition points and stability profiles.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To identify regions with temperature-dependent solvent accessibility changes.

• Site-specific labeling with environmentally sensitive fluorophores: Using probes like FlAsH to monitor conformational changes in specific helices and domains at different temperatures .

Based on studies of other A. aeolicus flagellar proteins, FliL likely adopts a more compact conformation at lower temperatures (20°C) and transitions to a more extended, functional state at physiological temperatures (85°C) .

How does the Na+-driven motor mechanism in A. aeolicus impact FliL function compared to H+-driven systems?

A. aeolicus utilizes a sodium-driven flagellar motor rather than the proton-driven motors found in many mesophilic bacteria . This distinction has significant implications for FliL function:

• Ion specificity: The interaction between FliL and the MotA/MotB stator complex may be optimized for Na+ coupling in A. aeolicus, potentially requiring different amino acid configurations at interaction surfaces.

• Thermostability considerations: Na+-driven motors may provide advantages at high temperatures, where proton gradients might be more difficult to maintain.

• Evolutionary significance: As A. aeolicus represents one of the earliest branches in bacterial evolution, its Na+-driven flagellar motor suggests that sodium coupling may have preceded proton coupling in evolutionary history .

Researchers investigating these differences should consider employing ion-substitution experiments, where Na+ is replaced with Li+ or other ions to evaluate motor function and FliL interaction specificity.

What insights can comparative genomics provide about the evolution of FliL in extremophiles?

Comparative genomic analysis of FliL across the bacterial phylogenetic tree reveals several important evolutionary patterns:

• Conservation of transmembrane domains: Despite sequence divergence, the transmembrane region topology is highly conserved, suggesting functional constraints .

• Variable phenotypic outcomes: The consequences of FliL deletion vary significantly between species, indicating adaptation to specific ecological niches .

• Co-evolution with stator components: Sequence analysis suggests co-evolutionary relationships between FliL and the MotA/MotB stator proteins, particularly in species adapted to extreme environments.

Research approaches should include phylogenetic analysis of FliL sequences, correlation of sequence features with growth temperature optima, and identification of conserved interaction motifs using tools like PRALINE and TMHMM .

How can researchers address solubility issues when working with recombinant A. aeolicus FliL?

The transmembrane domain of FliL presents significant challenges for solubility and purification. Evidence-based approaches to overcome these challenges include:

• Construct optimization: Creating truncated constructs (ΔfliL1) that retain functional domains while removing hydrophobic transmembrane regions .

• Fusion partners: Employing solubility-enhancing fusion tags such as MBP or SUMO, particularly effective for thermophilic proteins.

• Detergent screening: Systematic evaluation of detergents for membrane protein solubilization, with mild non-ionic detergents like DDM and LMNG often being effective for flagellar proteins.

• Expression temperature optimization: Lower induction temperatures (15-25°C) can increase soluble expression, despite being counterintuitive for thermophilic proteins.

The choice of approach should be guided by the intended experimental application, with structural studies typically requiring full-length protein in appropriate detergent environments, while functional assays may be successful with carefully designed truncation constructs.

What strategies can address the challenges of heterologous expression of A. aeolicus proteins in mesophilic hosts?

When expressing A. aeolicus FliL in mesophilic expression systems like E. coli, researchers encounter several challenges that can be addressed through specific interventions:

• Codon optimization: A. aeolicus has different codon usage patterns compared to E. coli, necessitating either codon-optimized synthetic genes or specialized E. coli strains supplying rare tRNAs.

• Protein folding: Chaperone co-expression (GroEL/GroES systems) can assist proper folding of thermophilic proteins at mesophilic temperatures.

• Functional assessment: When testing functionality through complementation, chimeric constructs combining domains from mesophilic and thermophilic homologs have proven more successful than direct expression of full A. aeolicus proteins .

• Host strain selection: The choice of E. coli strain significantly impacts expression outcomes, with BL21(DE3) derivatives often yielding better results for A. aeolicus proteins than K-12 derivatives.

These approaches have been validated through successful expression and functional characterization of other A. aeolicus flagellar components, including MotA and chimeric MotB constructs .

How does the function of FliL differ between thermophiles like A. aeolicus and mesophilic bacteria?

Comparative studies reveal distinct functional adaptations of FliL between thermophilic and mesophilic bacteria:

These differences reflect evolutionary adaptations to extreme environments and suggest that FliL function may be more specialized in thermophiles, potentially serving as a regulatory component rather than an essential structural element .

Can A. aeolicus FliL be utilized as a model system for understanding flagellar function in ancient bacteria?

Phylogenetic analyses position A. aeolicus as one of the earliest diverging bacterial lineages, making its flagellar components valuable for evolutionary studies . Key considerations for using A. aeolicus FliL as an evolutionary model include:

• Ancestral state reconstruction: Comparing A. aeolicus FliL sequences with diverse bacterial homologs can help infer ancestral protein states and evolutionary trajectories.

• Conservation of function: Despite sequence divergence, the negative modulatory role of FliL appears conserved across diverse species, suggesting an ancient functional role .

• Ion-specificity evolution: The Na⁺-driven motor in A. aeolicus likely represents the ancestral state, with H⁺-coupling evolving later in bacterial evolution .

• Thermoadaptation insights: Studying temperature-dependent conformational changes in A. aeolicus flagellar proteins provides insights into molecular adaptations enabling life in the high-temperature environments that may have characterized early Earth .

Researchers can leverage these evolutionary insights to design experiments investigating the fundamental principles of flagellar motor function and adaptation across diverse environmental conditions.

What emerging technologies could advance our understanding of A. aeolicus FliL structure and function?

Several cutting-edge approaches show promise for deepening our understanding of this thermophilic flagellar protein:

• Cryo-electron tomography: To visualize FliL in situ within the native flagellar apparatus, building on recent successes achieving near-atomic resolution (1.42 Å) with other A. aeolicus proteins .

• AlphaFold and related AI prediction tools: For generating structural models of full-length FliL and its interactions with other flagellar components.

• High-temperature single-molecule techniques: To observe FliL dynamics at physiologically relevant temperatures (85°C).

• CRISPR-based genetic manipulation: Development of genetic tools for direct manipulation of A. aeolicus would overcome a significant barrier to in vivo functional studies.

These approaches could help resolve outstanding questions about FliL's precise role in flagellar function and its adaptations for high-temperature environments.

How might insights from A. aeolicus FliL contribute to biotechnological applications?

Understanding the structure-function relationships in thermostable proteins like A. aeolicus FliL offers several potential biotechnological applications:

• Thermostable protein design: Principles derived from A. aeolicus proteins could inform the engineering of thermostable enzymes for industrial processes.

• Nanomotor development: The Na⁺-driven flagellar motor represents a natural nanomachine that functions efficiently at extreme temperatures, potentially inspiring biomimetic nanomotors.

• Biosensor applications: Temperature-responsive conformational changes observed in A. aeolicus flagellar proteins could be harnessed for designing biosensors with tunable response characteristics .

• Protein folding insights: The ability of A. aeolicus proteins to maintain function across wide temperature ranges provides valuable case studies for understanding protein folding and stability principles.

These applications demonstrate how fundamental research on extremophile proteins contributes to broader technological advances beyond basic scientific understanding.