Recombinant Thermus thermophilus Isoleucine--tRNA ligase (ileS), partial

What is the primary function of Thermus thermophilus IleRS?

Thermus thermophilus isoleucyl-tRNA synthetase (IleRS) is an enzyme that catalyzes the attachment of isoleucine to its corresponding tRNA (tRNA^Ile) in a two-step reaction. First, IleRS activates isoleucine with ATP to form isoleucyl-AMP. Second, it transfers the activated isoleucine to the 3'-end of tRNA^Ile to form Ile-tRNA^Ile. This aminoacylation reaction is crucial for protein biosynthesis, as it provides the charged tRNAs needed for translation .

Importantly, T. thermophilus IleRS possesses not only aminoacylation activity but also an editing function through its CP1 domain. This editing activity is essential because IleRS can mistakenly activate and transfer the structurally similar amino acid valine to tRNA^Ile. The CP1 domain specifically recognizes and deacylates the mischarged Val-tRNA^Ile, preventing translation errors that could lead to misfolded proteins .

How does the structure of T. thermophilus IleRS compare to IleRS from mesophilic organisms?

T. thermophilus IleRS shares the basic domain architecture with IleRS enzymes from mesophilic organisms but contains adaptations that contribute to its thermostability. Like other class I aminoacyl-tRNA synthetases, it contains a Rossmann fold catalytic domain, an editing domain (CP1), and additional domains involved in tRNA binding.

The crystal structure of the CP1 editing domain from T. thermophilus IleRS has been determined at high resolution (1.8 Å and 2.0 Å for the free and valine-bound forms, respectively), revealing the molecular basis for amino acid discrimination . In the complex structure, the Asp328 residue plays a critical role in recognizing the valine NH3+ group. The valine-binding pocket is precisely sized to accommodate valine but excludes isoleucine—the additional methylene group of isoleucine would clash with His319 .

Mutational analyses of the editing domain revealed the importance of multiple residues, including Thr228, Thr229, Thr230, and Asp328, which coordinate with water molecules in the editing active site . These structural features contribute to the high fidelity of amino acid selection in T. thermophilus IleRS.

What are the optimal expression systems for recombinant T. thermophilus IleRS?

For recombinant expression of T. thermophilus IleRS, Escherichia coli is typically the preferred host system due to its simplicity and high yield. The gene encoding T. thermophilus IleRS can be cloned into expression vectors such as pET-28b(+), which provides an N-terminal His-tag for simplified purification .

The expression methodology follows a standard protocol:

• Transform the expression construct into a suitable E. coli strain (e.g., BL21(DE3) or Rosetta).

• Grow transformants in rich media (e.g., LB or TB) with appropriate antibiotics.

• Induce protein expression with IPTG (typically 0.5-1 mM) when cultures reach mid-logarithmic phase.

• Grow cultures at a lower temperature (25-30°C) post-induction to enhance proper folding.

• Harvest cells by centrifugation and proceed with cell lysis and protein purification.

Alternative expression systems, such as the thermophilic bacterium T. thermophilus itself, can be considered for challenging constructs, though E. coli generally provides sufficient yield and proper folding for functional studies .

What purification protocol yields the highest activity for recombinant T. thermophilus IleRS?

A multi-step purification protocol is recommended to obtain high-purity, active T. thermophilus IleRS:

• Cell lysis: Disrupt cells using sonication or high-pressure homogenization in a buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 10 mM imidazole, and 5% glycerol.

• Immobilized metal affinity chromatography (IMAC): Apply the cleared lysate to a Ni-NTA column, wash with lysis buffer containing 20-30 mM imidazole, and elute the protein with 250-300 mM imidazole.

• Ion-exchange chromatography: Further purify the IMAC eluate using a Q-Sepharose column with a gradient of 50-500 mM NaCl in 20 mM Tris-HCl (pH 8.0).

• Size-exclusion chromatography: As a final polishing step, apply the protein to a Superdex 200 column equilibrated with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 5% glycerol.

Throughout the purification process, it's crucial to maintain a temperature of 4°C and include protease inhibitors in the buffers to prevent degradation. The purified enzyme can be stored in small aliquots at -80°C with 20-30% glycerol for long-term stability .

The specific activity of the purified enzyme should be assessed using an aminoacylation assay before use in experiments, as described in the biochemical characterization section.

How can the aminoacylation activity of T. thermophilus IleRS be measured?

The aminoacylation activity of T. thermophilus IleRS can be measured using several established methods:

• TLC-based aminoacylation assay: This method involves monitoring the incorporation of radiolabeled isoleucine ([³H] or [¹⁴C]) into tRNA. The reaction mixture typically contains purified IleRS, tRNA^Ile, ATP, isoleucine (including the radiolabeled fraction), and appropriate buffer components. After incubation at the desired temperature (often 55-65°C for T. thermophilus IleRS), aliquots are spotted onto TLC plates, and the aminoacylated tRNA is detected by autoradiography or scintillation counting .

• Pyrophosphate release assay: This continuous assay measures the release of pyrophosphate during the first step of the aminoacylation reaction. The pyrophosphate is coupled to the oxidation of NADH through a cascade of enzymatic reactions, allowing spectrophotometric monitoring at 340 nm.

• ATP-PP_i exchange assay: This assay measures the reversibility of the first step of aminoacylation by monitoring the incorporation of [³²P]PP_i into ATP.

For kinetic analysis, reactions should be performed under initial velocity conditions with varying concentrations of substrates (isoleucine, ATP, and tRNA^Ile) to determine K_M and k_cat values. The percentage of remaining IleRS activity in the presence of inhibitors can be calculated using the formula:

% remaining IleRS activity = (IleRS_wc × 100)/IleRS_nc

where IleRS_wc is the average of the IleRS activity measurements with compounds and IleRS_nc is the average of the IleRS activity measurements without compounds (vehicle only) .

What are the optimal conditions for T. thermophilus IleRS activity?

T. thermophilus IleRS exhibits maximum activity under specific conditions that reflect its adaptation to a thermophilic lifestyle:

Temperature: The optimal temperature for T. thermophilus IleRS activity is approximately 65°C, significantly higher than the 37°C optimum of mesophilic enzymes. The enzyme maintains considerable activity between 55-75°C and exhibits remarkable thermostability, retaining activity even after prolonged incubation at elevated temperatures .

pH: The optimal pH for T. thermophilus IleRS activity is around 8.0, similar to other thermostable enzymes from Thermus species .

Buffer composition: A typical reaction buffer contains:

• 50 mM HEPES or Tris-HCl (pH 8.0)

• 10 mM MgCl₂ (essential for ATP binding)

• 50 mM KCl

• 1 mM DTT (to maintain reduced cysteines)

• 0.1 mg/ml BSA (to stabilize the enzyme)

Ionic strength: Moderate ionic strength (50-100 mM KCl or NaCl) generally supports optimal activity, with higher salt concentrations potentially inhibitory.

Substrates: For steady-state kinetic measurements, the following substrate concentrations are typically used:

• ATP: 2-5 mM

• L-isoleucine: 50-100 μM

• tRNA^Ile: 2-5 μM

Due to its thermostability, T. thermophilus IleRS allows for extended reaction times and elevated temperatures, which can be advantageous for certain applications requiring robust enzymatic activity .

How can the CP1 editing domain of T. thermophilus IleRS be crystallized for structural studies?

The CP1 editing domain of T. thermophilus IleRS has been successfully crystallized for high-resolution structural analysis using the following approach:

• Domain expression and purification: Clone the CP1 domain (typically residues ~185-405) into an expression vector with an affinity tag. Express in E. coli and purify using standard chromatography techniques including affinity, ion-exchange, and size-exclusion chromatography.

• Initial crystallization screening: Perform sparse matrix screening at different protein concentrations (typically 5-15 mg/ml) using commercial crystallization kits. Include both apo-protein and protein-ligand complexes (with valine or analogs) in the screening.

• Optimization: Refine promising crystallization conditions by varying precipitant concentration, pH, temperature, and additives. For the T. thermophilus IleRS CP1 domain, successful crystals have been obtained using:

  • Hanging-drop vapor diffusion method

  • Protein concentration of 10 mg/ml

  • Reservoir solution containing 100 mM HEPES (pH 7.5), 200 mM MgCl₂, and 30% (w/v) PEG 4000

  • Temperature of 20°C

  • Drop size of 1 μl protein + 1 μl reservoir solution

• Co-crystallization with ligands: For the valine-bound structure, co-crystallize the domain with 5-10 mM L-valine added to the protein solution or soak apo-crystals in mother liquor containing the ligand.

• Cryoprotection: Prior to data collection, briefly soak crystals in a cryoprotectant solution (reservoir solution supplemented with 15-20% glycerol or ethylene glycol) and flash-freeze in liquid nitrogen.

Data collection at a synchrotron source allowed the determination of the T. thermophilus IleRS CP1 domain structure at 1.8 Å resolution for the apo form and 2.0 Å for the valine complex, revealing the molecular details of substrate recognition and discrimination .

What mutagenesis approaches are effective for studying the editing mechanism of T. thermophilus IleRS?

Site-directed mutagenesis provides valuable insights into the editing mechanism of T. thermophilus IleRS. Effective approaches include:

• Alanine scanning mutagenesis: Systematically replace conserved residues in the editing domain with alanine to identify those critical for editing activity. Key residues that have been identified in the T. thermophilus IleRS CP1 domain include Thr228, Thr229, Thr230, and Asp328, which are involved in substrate recognition and catalysis .

• Conservative substitutions: Replace residues with chemically similar amino acids to probe the importance of specific chemical properties. For example, Asp328 could be mutated to Glu to determine if the negative charge is sufficient or if the precise positioning is essential.

• Cross-species mutations: Introduce mutations based on corresponding residues in other species' IleRS enzymes. For instance, the H319A mutation in E. coli IleRS causes deacylation of both Val-tRNA^Ile and Ile-tRNA^Ile, suggesting a critical role in substrate discrimination .

• Domain swapping: Exchange the CP1 domain between IleRS and other related tRNA synthetases (e.g., ValRS or LeuRS) to investigate domain-specific contributions to editing specificity.

To assess the effects of mutations, compare the following activities between wild-type and mutant enzymes:

• Aminoacylation activity: Determine if the mutation affects the synthetic function using standard aminoacylation assays.

• Post-transfer editing activity: Measure the ability to deacylate mischarged Val-tRNA^Ile using pre-charged substrates.

• Pre-transfer editing activity: Assess ATP hydrolysis in the presence of non-cognate amino acids.

• Binding affinity: Use isothermal titration calorimetry or surface plasmon resonance to measure binding constants for various substrates.

Through these approaches, researchers have determined that His319 in T. thermophilus IleRS creates a steric gate that prevents isoleucine binding in the editing site, while Asp328 forms critical interactions with the amino group of the valine substrate .

How can T. thermophilus IleRS be used as a model for studying enzyme thermostability?

T. thermophilus IleRS serves as an excellent model for investigating enzyme thermostability due to its natural adaptation to extreme temperatures. Researchers can utilize the following approaches to study thermostability mechanisms:

• Comparative structural analysis: Compare the crystal structures of T. thermophilus IleRS with mesophilic homologs (e.g., E. coli IleRS) to identify structural features contributing to thermostability, such as:

  • Increased number of salt bridges and hydrogen bonds

  • Higher proportion of charged residues on the protein surface

  • Reduced number of thermolabile residues (Asn, Gln, Cys, Met)

  • More compact protein core with optimized hydrophobic interactions

  • Shorter loops and enhanced secondary structure elements

• Thermal denaturation studies: Measure protein unfolding using differential scanning calorimetry (DSC) or circular dichroism (CD) spectroscopy at different temperatures to determine the melting temperature (Tm) and thermodynamic parameters of stability. For T. thermophilus IleRS, these experiments should be conducted across a wide temperature range (25-100°C) to capture the complete unfolding profile.

• Activity retention assays: Assess the enzyme's aminoacylation activity after incubation at elevated temperatures (65-95°C) for various time periods. The T. thermophilus enzyme typically maintains significant activity even after prolonged heating, unlike its mesophilic counterparts .

• Chimeric enzyme construction: Create hybrid enzymes by swapping domains between thermophilic and mesophilic IleRS variants to identify regions that contribute most significantly to thermostability.

• Directed evolution approaches: Implement laboratory evolution strategies to enhance or modify the thermostability properties, providing insights into the sequence-stability relationship.

By applying these methodologies, researchers have demonstrated that T. thermophilus IleRS maintains its editing function at high temperatures without compromising specificity, indicating a finely balanced adaptation of both catalytic efficiency and structural integrity to extreme conditions .

Can T. thermophilus IleRS be used to study the evolution of editing mechanisms in aminoacyl-tRNA synthetases?

T. thermophilus IleRS provides an excellent system for investigating the evolution of editing mechanisms in aminoacyl-tRNA synthetases through several experimental approaches:

• Phylogenetic analysis: Compare IleRS sequences across the tree of life, from thermophilic archaea and bacteria to mesophilic and eukaryotic organisms. The conservation of editing domain residues across diverse species indicates their fundamental importance in maintaining translational fidelity throughout evolution.

• Structure-function comparisons: Analyze the structural conservation of editing domains across different classes of aminoacyl-tRNA synthetases. The CP1 domain of T. thermophilus IleRS can be compared with editing domains from related synthetases like ValRS and LeuRS to identify common ancestral features and specialized adaptations .

• Ancestral sequence reconstruction: Computationally predict and experimentally test ancestral IleRS sequences to trace the evolutionary trajectory of editing mechanisms. This approach can reveal whether high-temperature environments influenced the development of editing capabilities.

• Cross-species complementation studies: Test whether the T. thermophilus IleRS can functionally replace IleRS in mesophilic organisms and vice versa, particularly focusing on editing function maintenance across different cellular environments.

• Comparative kinetic analysis: Measure the editing efficiency against various non-cognate amino acids in IleRS enzymes from different sources. The T. thermophilus enzyme shows remarkably precise discrimination between isoleucine and valine despite functioning at elevated temperatures .

Specific experiments have revealed that the editing mechanism in T. thermophilus IleRS differs in some aspects from that of the T. thermophilus leucyl-tRNA synthetase. For example, the carbonyl oxygens of the amino acids are positioned on opposite sides of the adenosine ribose ring and are recognized through different molecular interactions . These differences highlight the independent evolutionary paths these related enzymes have taken while maintaining their essential editing functions.

How do mutations in the CP1 domain affect the fidelity of T. thermophilus IleRS?

Mutations in the CP1 domain of T. thermophilus IleRS can dramatically affect the enzyme's fidelity through various mechanisms, making this a rich area for advanced research. The effects can be analyzed at multiple levels:

Research has shown that mutations in the T. thermophilus IleRS CP1 domain can create variants with altered substrate specificity profiles, demonstrating that the editing mechanism represents a finely tuned evolutionary compromise between efficient aminoacylation and error correction .

What are the molecular mechanisms of thermostability in T. thermophilus IleRS compared to mesophilic homologs?

The molecular mechanisms underlying the remarkable thermostability of T. thermophilus IleRS involve multiple structural and biophysical adaptations that can be investigated through advanced experimental approaches:

The thermostability mechanisms of T. thermophilus IleRS likely share commonalities with other thermostable proteins from this organism, such as DNA ligase, which maintains activity even at 65°C and shows strong thermostability compared to mesophilic homologs . This suggests that adaptation to high-temperature environments involves coherent changes across multiple cellular systems.

Understanding these molecular mechanisms not only provides fundamental insights into protein thermostability but also informs protein engineering efforts aimed at creating thermostable variants for biotechnological applications.

What are the emerging applications of recombinant T. thermophilus IleRS in research and biotechnology?

Recombinant T. thermophilus IleRS has several emerging applications in both fundamental research and biotechnology:

• Thermostable cell-free protein synthesis systems: The thermostability of T. thermophilus IleRS makes it valuable for developing high-temperature cell-free translation systems, which offer advantages including reduced contamination risk, increased reaction rates, and improved folding of certain proteins. Incorporating thermostable IleRS alongside other translation components from thermophilic organisms can create robust systems operating at 50-65°C .

• Drug discovery platforms: As demonstrated with Trypanosoma brucei, IleRS can serve as a drug target for developing novel antimicrobials . The T. thermophilus enzyme provides a stable, well-characterized model system for structure-based drug design, particularly for screening compounds targeting the editing domain.

• Protein engineering applications: The detailed structural understanding of T. thermophilus IleRS, particularly its editing domain, enables rational design of variants with altered substrate specificity or enhanced thermostability. These engineered enzymes could have applications in:

  • Incorporating non-canonical amino acids into proteins

  • Developing biosensors for amino acid detection

  • Creating orthogonal translation systems

• Evolutionary studies: The well-characterized T. thermophilus IleRS serves as a model for understanding molecular adaptation to extreme environments and the co-evolution of editing mechanisms and translation fidelity.

• Educational tools: Due to its stability and well-documented structure-function relationships, T. thermophilus IleRS is an excellent model system for teaching concepts in enzyme kinetics, protein structure, and molecular evolution.

As research continues to advance, the development of engineered T. thermophilus IleRS variants with superior properties through directed evolution approaches will likely expand its applications in synthetic biology and biotechnology .

What are the current knowledge gaps and future research directions for T. thermophilus IleRS?

Despite significant advances in understanding T. thermophilus IleRS, several knowledge gaps and promising research directions remain:

• Structural dynamics during catalysis: While static crystal structures of the CP1 domain have been determined , the dynamic conformational changes during substrate binding, catalysis, and product release remain incompletely understood. Future research using techniques such as single-molecule FRET, time-resolved crystallography, or cryo-electron microscopy could provide insights into these transient states.

• Complete structural characterization: Crystal structures of the full-length T. thermophilus IleRS in complex with tRNA and during different stages of the aminoacylation and editing reactions would provide a comprehensive understanding of the enzyme's mechanism.

• Interactions with translation machinery: Studies exploring how T. thermophilus IleRS interacts with other components of the translation apparatus (ribosomes, elongation factors) at high temperatures could reveal adaptations in the broader translation system.

• Comparative studies with dual IleRS systems: Some bacteria possess two types of IleRS genes (ileS1 and ileS2) with different regulation patterns and properties . Comparative studies between T. thermophilus IleRS and such dual systems could provide insights into the evolution and specialization of these enzymes.

• Development of orthogonal systems: Engineering T. thermophilus IleRS variants that can specifically charge tRNAs with non-canonical amino acids while maintaining thermostability would advance applications in synthetic biology.

• Mechanism of thermal adaptation: While general features contributing to thermostability have been identified, the precise molecular mechanisms and evolutionary pathways by which T. thermophilus IleRS acquired its thermostability remain to be fully elucidated.

• Applied research directions: Similar to advances with thermostable DNA ligases , research into creating T. thermophilus IleRS mutants with enhanced properties (higher thermostability, altered substrate specificity, or improved catalytic efficiency) through directed evolution approaches could yield variants with valuable biotechnological applications.