Recombinant Sulfolobus solfataricus Phenylalanine--tRNA ligase beta subunit (pheT), partial

What is the molecular structure of Sulfolobus solfataricus PheRS?

Sulfolobus solfataricus PheRS is a heterotetramic enzyme composed of two different subunits: alpha (56 kDa) and beta (64 kDa). This structure represents the alpha2beta2 configuration typical of archaeal and eukaryotic PheRS enzymes rather than the alpha2 configuration found in most bacterial species. The enzyme functions optimally under extreme conditions, reflecting S. solfataricus' natural environment of high temperature (around 80°C) and acidic pH (2-4). The beta subunit (PheT) contains domains responsible for tRNA binding and plays a crucial role in the enzyme's substrate specificity and catalytic efficiency .

How does S. solfataricus PheT compare to its counterparts in bacteria and eukaryotes?

Research reveals that S. solfataricus PheT shares greater structural and functional similarities with eukaryotic phenylalanyl-tRNA synthetases than with bacterial homologs. Both archaeal and eukaryotic PheRS exist as heterotetramers with distinct alpha and beta subunits, while most bacterial enzymes are homodimers (alpha2). Functionally, S. solfataricus PheT demonstrates tRNA specificity patterns more similar to eukaryotic systems, showing high substrate discrimination and interacting with elongation factor 1alpha to enhance aminoacylation efficiency . This similarity supports the evolutionary relationship between archaea and eukaryotes in the context of translation machinery components.

What are the unique adaptations of S. solfataricus PheT for extreme environments?

S. solfataricus PheT exhibits several structural adaptations that allow it to function under extreme conditions including temperatures around 80°C and pH levels between 2-4. These adaptations likely include increased surface charge, enhanced hydrophobic interactions, additional salt bridges, and a more compact protein core. Unlike mesophilic counterparts, the archaeal PheT maintains structural integrity and catalytic function under conditions that would denature most proteins. This thermostability makes it particularly valuable for in vitro applications requiring high-temperature reactions .

What expression systems are most effective for producing recombinant S. solfataricus PheT?

Several expression systems have been developed for S. solfataricus proteins, with varying efficiency. For homologous expression, virus-based shuttle vectors have proven effective, particularly those derived from SSV1 with selectable markers like pyrEF for complementation of uracil auxotrophic mutants. These systems allow for stable propagation under selective conditions and can achieve strong, heat-inducible expression . For heterologous expression in E. coli, specialized vectors incorporating thermostable tags and chaperones can improve folding and solubility of archaeal proteins. When expressing PheT specifically, codon optimization for the host organism and inclusion of a C-terminal six-His tag facilitates purification while maintaining activity .

How can researchers overcome challenges in purifying functional recombinant PheT?

Purification of active S. solfataricus PheT presents several challenges that can be addressed through methodological refinements:

Additionally, researchers should consider using affinity chromatography with nickel columns for His-tagged proteins, followed by ion exchange and size exclusion chromatography to achieve high purity while maintaining functional integrity .

What are the critical factors for reconstituting active S. solfataricus PheRS from recombinant subunits?

Reconstitution of active S. solfataricus PheRS requires careful consideration of both alpha and beta subunit expression and assembly. The tetrameric structure (alpha2beta2) must be correctly formed, with proper stoichiometry between subunits. Research indicates that co-expression of both subunits in the same host often yields better results than separate expression and in vitro reconstitution. If expressing separately, the reconstitution buffer should contain stabilizing agents such as glycerol (10-15%) and reducing agents to maintain proper disulfide bonding. The assembly process is temperature-dependent, with optimal reconstitution occurring at moderate temperatures (40-50°C) followed by a thermal activation step at higher temperatures (70-80°C). Additionally, the presence of substrate tRNA during reconstitution can enhance the formation of catalytically active complexes .

What methods are most reliable for assessing S. solfataricus PheT activity in vitro?

The aminoacylation assay remains the gold standard for measuring PheT activity, with several methodological variations applicable to S. solfataricus enzyme:

• Radioactive assay: Using [14C] or [3H]-labeled phenylalanine to measure tRNA aminoacylation rates through filter binding and scintillation counting. This method provides high sensitivity but requires radioisotope handling capabilities.

• ATP-PPi exchange assay: Measures the reverse reaction catalyzed by PheRS, providing insights into amino acid activation independent of tRNA charging.

• HPLC-based assays: Separate and quantify charged vs. uncharged tRNA species, offering a non-radioactive alternative with good sensitivity.

For thermostable enzymes like S. solfataricus PheT, assay conditions must be carefully controlled with reactions performed at elevated temperatures (typically 65-80°C) and buffers resistant to pH shifts at high temperatures (e.g., HEPES or MES rather than Tris). Importantly, researchers must use endogenous S. solfataricus tRNA or verified compatible tRNAs, as the enzyme shows strict specificity for archaeal tRNAPhe .

How does temperature affect the kinetics and fidelity of S. solfataricus PheT?

Temperature exerts complex effects on S. solfataricus PheT kinetics and fidelity that differ from mesophilic enzymes:

At optimal temperatures (70-85°C), S. solfataricus PheT demonstrates enhanced substrate binding and catalytic efficiency while maintaining high fidelity in discriminating against non-cognate amino acids. This temperature-dependent modulation of activity involves conformational changes that optimize active site geometry and substrate interactions. Researchers examining kinetics must account for these temperature effects when comparing archaeal PheT with mesophilic counterparts .

What is the mechanism of S. solfataricus PheT interaction with elongation factor 1alpha?

S. solfataricus PheT has been shown to interact with elongation factor 1alpha (EF-1α), significantly enhancing tRNA aminoacylation efficiency. This interaction represents a form of tRNA channeling mechanism similar to that observed in eukaryotic systems. The binding appears to facilitate the transfer of charged tRNA from PheRS to EF-1α, optimizing the translation process under extreme conditions. The interaction involves specific binding domains on the beta subunit (PheT) that recognize complementary surfaces on EF-1α. This interaction is strengthened at elevated temperatures and may represent an adaptation for maintaining translation efficiency under extreme conditions. When designing experiments to study PheT function, researchers should consider the potential role of EF-1α in modulating activity, particularly when assessing in vivo function or reconstituting translation systems .

What genetic tools are available for manipulating S. solfataricus to study PheT in vivo?

Several genetic tools have been developed for S. solfataricus that allow in vivo studies of proteins like PheT:

• Shuttle vectors: Plasmids capable of replication in both E. coli and S. solfataricus, including those based on SSV1 virus that can be maintained under selective conditions using pyrEF complementation in uracil auxotrophic mutants.

• Selectable markers: Besides pyrEF for uracil auxotrophy complementation, systems based on β-galactosidase (lacS) complementation allow blue/white screening using X-β-Gal.

• Expression systems: The T6 promoter from SSV1 provides high transcription rates and can be used to drive expression of recombinant proteins.

• Homologous recombination systems: Allow for chromosomal integration and gene replacement, enabling the creation of knockout or modification strains.

When designing experiments to study PheT in vivo, researchers should consider using these tools in combination with temperature-inducible promoters to control expression levels. The construction of tagged variants (e.g., with C-terminal His-tags) facilitates downstream purification and detection while enabling protein-protein interaction studies under native conditions .

How can researchers design effective mutagenesis strategies for S. solfataricus PheT?

Effective mutagenesis of S. solfataricus PheT requires specialized approaches that account for the challenges of working with archaeal proteins:

The mutant proteins should be characterized through activity assays performed at different temperatures (50-90°C) to assess both catalytic parameters and thermal stability profiles .

What are the challenges of expressing S. solfataricus PheT in heterologous systems?

Expressing S. solfataricus PheT in heterologous systems presents several challenges that must be addressed for successful production:

For the best results with S. solfataricus PheT, researchers often use a combination of approaches, such as expressing the protein with solubility-enhancing fusion partners in protease-deficient strains at reduced temperatures (15-25°C). Alternative heterologous hosts like Sulfolobus acidocaldarius might provide better expression of functional protein compared to E. coli due to more compatible cellular machinery .

How can S. solfataricus PheT be utilized in in vitro protein synthesis systems?

S. solfataricus PheT offers unique advantages for in vitro protein synthesis systems designed to operate at elevated temperatures:

• Thermostable translation components: PheT can be incorporated into completely thermostable in vitro translation systems capable of functioning at 65-75°C, conditions that inactivate most mesophilic proteins.

• Extended reaction times: The exceptional stability of S. solfataricus PheT allows for prolonged aminoacylation reactions, potentially increasing protein yield in cell-free systems.

• Increased reaction kinetics: Higher operating temperatures accelerate reaction rates, potentially improving the efficiency of in vitro protein synthesis.

• Reduced contamination risk: Elevated temperatures inhibit growth of mesophilic contaminants, offering advantages for long-running cell-free reactions.

• Enhanced folding of thermostable proteins: In vitro systems incorporating archaeal components may improve folding of thermophilic target proteins by operating at their native temperatures.

When implementing such systems, researchers must ensure all components (ribosomes, translation factors, synthetases) derive from thermophilic sources or are engineered for thermostability. The advantage of S. solfataricus PheT is its natural adaptation to extreme conditions, eliminating the need for extensive engineering .

What are the emerging techniques for studying PheT-tRNA interactions at high temperatures?

Several advanced techniques have been adapted or developed specifically for studying PheT-tRNA interactions under extreme temperature conditions:

• Thermostable fluorescent labeling: Modified fluorophores with enhanced thermal stability can be attached to tRNA or PheT for real-time monitoring of interactions at elevated temperatures.

• Surface Plasmon Resonance (SPR) with thermostable chips: Specialized SPR systems capable of operating at 60-80°C allow for kinetic analysis of PheT-tRNA binding.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) at high temperatures: Reveals conformational dynamics and binding interfaces under near-native conditions for thermophilic proteins.

• Cryo-electron microscopy (cryo-EM) with rapid thermal quenching: Captures structural states of PheT-tRNA complexes after incubation at high temperatures by ultra-fast cooling.

• Molecular dynamics simulations parameterized for high temperatures: Computational approaches specifically designed to model biomolecular interactions at extreme temperatures.

These techniques provide complementary information about the structural and kinetic aspects of PheT-tRNA interactions under conditions that mimic the natural environment of S. solfataricus .

How does PheT from S. solfataricus contribute to our understanding of the evolution of translation machinery?

S. solfataricus PheT offers valuable insights into the evolution of translation machinery across domains of life:

• Archaeal-eukaryotic similarities: The heterotetramic structure (α2β2) and functional characteristics of archaeal PheRS more closely resemble eukaryotic systems than bacterial counterparts, supporting the evolutionary relationship between Archaea and Eukarya.

• Adaptation mechanisms: Comparing thermostable archaeal PheT with mesophilic homologs reveals evolutionary strategies for protein adaptation to extreme environments without compromising essential functions.

• Ancient conserved features: Core catalytic domains of PheT that remain unchanged across all domains of life likely represent ancient, essential features of the translation apparatus that evolved before the divergence of the three domains.

• Domain-specific innovations: Features unique to archaeal PheT, particularly those related to thermostability or acidophily, illustrate domain-specific adaptations that emerged after evolutionary divergence.

• tRNA recognition patterns: The specificity of S. solfataricus PheT for archaeal tRNAPhe provides insights into the co-evolution of synthetases and their cognate tRNAs.

Research on S. solfataricus PheT supports the hypothesis that modern protein synthesis machinery evolved from a thermophilic last universal common ancestor (LUCA), with archaeal systems potentially representing the most conserved version of the ancient translation apparatus .

What are the most common issues encountered when working with recombinant S. solfataricus PheT?

Researchers frequently encounter several challenges when working with recombinant S. solfataricus PheT:

• Loss of activity during purification: This often results from inappropriate buffer conditions or exposure to inhibitory compounds. Maintain reducing conditions throughout purification and include stabilizing agents like glycerol or specific ions (Mg2+, Zn2+) known to support PheT structure.

• Inconsistent aminoacylation assay results: Variable activity can stem from batch-to-batch differences in tRNA preparation. Use consistently prepared tRNA stocks and include internal standards in each assay.

• Protein aggregation after purification: Common during concentration or storage. Add non-ionic detergents (0.01-0.05% Triton X-100) or increase salt concentration to maintain solubility.

• Low expression yields: Often due to toxicity or codon usage issues. Optimize expression conditions by testing different hosts, promoters, and induction parameters, or consider using archaeal expression systems.

• Difficulty distinguishing alpha and beta subunits: Both subunits have similar molecular weights. Use tagged versions of one subunit or apply specialized separation techniques like ion-exchange chromatography .

How can researchers distinguish between structural and functional effects of PheT mutations?

Distinguishing structural from functional effects of PheT mutations requires a multi-faceted experimental approach:

• Thermal stability assays: Techniques such as differential scanning fluorimetry (DSF) or circular dichroism (CD) spectroscopy can reveal whether mutations affect protein folding or stability independent of catalytic function.

• Limited proteolysis: Comparing digestion patterns of wild-type and mutant proteins can identify structural perturbations that alter protease accessibility.

• Activity assays with varying substrates: Testing activity with different tRNA species or amino acid analogs can distinguish between effects on substrate binding versus catalysis.

• Binding assays independent of catalysis: Surface plasmon resonance or fluorescence anisotropy measurements of tRNA binding can separate binding effects from catalytic defects.

• Structural analysis: When possible, obtain crystal structures or cryo-EM models of mutant proteins to directly visualize structural changes.

For thermostable proteins like S. solfataricus PheT, these analyses should be performed at multiple temperatures to distinguish temperature-dependent effects from general structural or functional impairments .

What strategies can overcome low yields of functional recombinant S. solfataricus PheT?

Several strategies can address the challenge of low yields when producing functional recombinant S. solfataricus PheT:

When implementing these strategies, researchers should optimize one variable at a time and quantify both total and functional protein yields to identify the most effective approach for their specific experimental needs .

What are promising avenues for engineering S. solfataricus PheT for biotechnology applications?

Several promising research directions for engineering S. solfataricus PheT include:

• Enhanced thermostability engineering: Further increasing the temperature range of PheT activity could enable novel high-temperature biotechnology applications, particularly in PCR-like reactions requiring repeated thermal cycling.

• Substrate specificity modification: Engineering PheT to incorporate non-canonical amino acids could enable the synthesis of novel proteins with unique properties under extreme conditions.

• Solvent tolerance enhancement: Modifying the enzyme to function in organic solvents while maintaining thermostability could create biocatalysts for industrial processes requiring both high temperatures and non-aqueous environments.

• Immobilization strategies: Developing methods for stable immobilization of PheT on various supports could enable continuous aminoacylation processes for biotechnology applications.

• Fusion with other thermostable enzymes: Creating bifunctional enzymes by fusing PheT with other thermostable proteins could enable novel cascade reactions under extreme conditions.

These engineering efforts should consider the unique structural features of archaeal PheT that contribute to its thermostability while preserving essential catalytic functions .

How might structural studies of S. solfataricus PheT advance our understanding of archaeal translation?

Detailed structural studies of S. solfataricus PheT could significantly advance our understanding of archaeal translation through several avenues:

• High-resolution structures of the complete heterotetramer in different functional states would reveal conformational changes during the aminoacylation cycle.

• Co-crystal structures with tRNA and amino acid substrates would illuminate the molecular basis for substrate recognition and catalysis under extreme conditions.

• Structures of PheT-EF-1α complexes would provide insight into the tRNA channeling mechanism that enhances translation efficiency in archaea.

• Comparative structural analysis across temperature ranges could reveal dynamic aspects of thermoadaptation in translation machinery.

• Identification of archaeal-specific structural features might provide targets for developing selective inhibitors for research or potential antimicrobial applications against pathogenic archaea.

Modern techniques like cryo-EM are particularly well-suited for these studies, as they can capture the enzyme in various functional states without the constraints of crystal packing .

What is the potential for using S. solfataricus PheT as a model for understanding disease-associated mutations in human PheRS?

S. solfataricus PheT serves as a valuable model for understanding disease-associated mutations in human PheRS due to several factors:

• Conserved core architecture: Despite evolutionary distance, the core domains and catalytic mechanisms are highly conserved between archaeal and human PheRS.

• Simplified experimental system: The thermostability of S. solfataricus PheT facilitates structural and biochemical studies that might be challenging with the more complex human enzyme.

• Structure-function correlations: Mutations equivalent to human disease variants can be introduced into archaeal PheT and analyzed under varying conditions to determine their effects on enzyme function.

• Evolutionary insights: Comparing archaeal and human PheRS responses to equivalent mutations can reveal which functional aspects are most evolutionarily constrained, potentially indicating their clinical significance.

• Drug development platform: Archaeal PheT can serve as a simplified screening platform for compounds targeting conserved active sites, potentially leading to therapies for disorders caused by PheRS dysfunction.

Researchers investigating human PheRS-related diseases should consider creating equivalent mutations in S. solfataricus PheT to gain mechanistic insights into pathogenicity while benefiting from the experimental advantages of working with a thermostable archaeal protein .