Recombinant Thermus thermophilus Elongation factor Ts (tsf)

What is the structural organization of the Thermus thermophilus EF-Ts gene and how does it compare to other bacterial species?

The gene encoding Elongation Factor Ts from Thermus thermophilus shares similar structural organization to that found in Escherichia coli, with the ribosomal protein S2 gene located at the 5' end . When comparing the amino acid sequence, T. thermophilus EF-Ts shows considerable homology with other bacterial EF-Ts proteins, exhibiting approximately 44% sequence identity with Thermus thermophilus and 68% with E. coli . Notably, the T. thermophilus EF-Ts is considerably shorter than its E. coli counterpart, differing by 86 amino acids . This structural variation may contribute to its unique thermostability properties while maintaining functional conservation.

What are the key structural features of purified recombinant T. thermophilus EF-Ts?

Purified recombinant T. thermophilus EF-Ts exists as a homodimer in solution, stabilized by:

• A disulfide bridge between two cysteine residues at position 190 (Cys190) of each monomer

• An extensive dimer interface formed by a three-stranded antiparallel β-sheet from each subunit that interact to form a β-sandwich

• Several hydrophobic interactions, particularly involving residues Leu73, Cys190, and Phe192 that form a hydrophobic core at the dimerization interface

Unlike the predominantly α-helical interface that stabilizes the E. coli EF-Ts dimer, the T. thermophilus EF-Ts dimer is characterized by this distinct β-sandwich structure . This dimerization significantly contributes to the thermostability of T. thermophilus EF-Ts and represents a potential adaptation strategy of the translation system in this thermophile to withstand high temperatures .

What is the functional significance of T. thermophilus EF-Ts dimerization, and how can this be experimentally verified?

Dimerization of T. thermophilus EF-Ts is critical for its function as a nucleotide exchange factor. Studies have demonstrated that EF-Ts variants unable to form dimers were also inactive in facilitating nucleotide exchange on EF-Tu . This can be experimentally verified through:

• Site-directed mutagenesis: Introducing mutations in the dimerization interface (e.g., replacing Leu73, Cys190, or Phe192 with amino acids like Asp or Ala) disrupts dimer formation without affecting the tertiary structure of individual subunits .

• Functional assays: Measuring nucleotide exchange rates (GDP/GTP exchange) on EF-Tu using wild-type and mutant EF-Ts variants.

• Structural verification: Using techniques like gel permeation chromatography, polyacrylamide gel electrophoresis, and CD spectroscopy to confirm that the loss of activity is not due to changes in secondary structure but rather to the inability to form dimers .

How do the quaternary structure complexes of T. thermophilus EF-Ts with EF-Tu differ from those of mesophilic bacteria?

T. thermophilus EF-Ts forms specific quaternary structure complexes with EF-Tu that differ from mesophilic bacteria:

• Quaternary (EF-Tu·EF-Ts)₂ complex: This heterotetramer consists of two EF-Tu molecules bound to the EF-Ts dimer .

• Ternary EF-Tu·EF-Ts₂ complex: This complex has also been detected by gel permeation chromatography and polyacrylamide gel electrophoresis, suggesting alternative binding modes between EF-Tu and the EF-Ts dimer .

These quaternary structures contrast with the simpler binary EF-Tu·EF-Ts complexes typically observed in mesophilic bacteria. The requirement for T. thermophilus EF-Ts to function as a homodimer indicates an adaptation in the thermophilic translation machinery . The formation of these higher-order complexes likely contributes to the stability and efficiency of the nucleotide exchange process at elevated temperatures, representing an evolutionary strategy to maintain translation fidelity under extreme conditions.

What methodologies are most effective for studying the nucleotide exchange activity of recombinant T. thermophilus EF-Ts?

For studying nucleotide exchange activity of recombinant T. thermophilus EF-Ts, researchers can employ several methodologies:

• Radioisotope-based exchange assays: Monitoring the dissociation rate of [³H]GDP from preformed EF-Tu·[³H]GDP complex in the presence of EF-Ts. Studies with psychrophilic EF-Ts showed that even at very low Tu:Ts ratios, the exchange rate can be enhanced by orders of magnitude .

• Kinetic measurements: Determining the energy of activation (Ea) of the exchange reaction to understand how EF-Ts lowers the activation barrier for GDP dissociation from EF-Tu .

• Temperature-dependent activity assays: Conducting exchange experiments at different temperatures (from 37°C to 65°C or higher) to assess the thermostability and optimal temperature range for recombinant T. thermophilus EF-Ts activity .

• Fluorescence-based approaches: Using fluorescently labeled nucleotides to monitor exchange kinetics in real-time without radioactivity.

• Reconstituted translation systems: For functional validation, incorporating purified EF-Ts into a reconstituted T. thermophilus translation system containing ribosomes, tRNAs, and other translation factors to assess its activity in a more complete biological context .

What are the critical factors to consider when designing experiments to analyze the thermostability of T. thermophilus EF-Ts?

When analyzing thermostability of T. thermophilus EF-Ts, researchers should consider:

• Buffer composition: The specific buffer components significantly affect thermostability. Optimal conditions for T. thermophilus translation components typically include:

  • 50 mM HEPES-KOH, pH 8

  • 100 mM K-Glutamate

  • 10 mM Mg(OAc)₂

  • Polyamines (e.g., 2 mM spermine) which are required for translation activity at both high and low temperatures

• Calorimetric measurements: Differential scanning calorimetry should be employed to quantitatively assess how dimerization contributes to thermostability .

• Mutation design strategy: When performing site-directed mutagenesis to study thermostability determinants, carefully consider:

  • Residues involved in the dimer interface (Leu73, Cys190, Phe192)

  • Mutations that disrupt dimerization without affecting secondary structure

• CD spectroscopy: This technique can confirm that changes in activity following mutations are not due to alterations in protein secondary structure .

• Concentration effects: The concentration of EF-Ts can affect dimerization equilibrium and subsequently thermostability properties, so concentration must be carefully controlled in experimental designs.

How can recombinant T. thermophilus EF-Ts be effectively incorporated into a reconstituted cell-free protein synthesis system?

For effective incorporation into reconstituted cell-free protein synthesis systems, consider the following methodology:

• Component preparation:

  • Purify ribosomes, transfer RNAs (tRNAs), and recombinant translation factors including EF-Ts from T. thermophilus

  • Ensure all components maintain native structure and activity through appropriate purification and storage conditions

• Optimal buffer composition for thermophilic translation:

  • 50 mM HEPES-KOH, pH 8

  • 100 mM K-Glutamate

  • 10 mM Mg(OAc)₂

  • 2 mM spermine

  • 7.2 mM DTT

  • 2 mM ATP, 2 mM GTP

  • 0.02 mM N¹⁰-formyl-tetrahydrofolate

  • 15 mM phospho(enol)pyruvic acid

  • Appropriate concentrations of amino acids

  • 2 mg/ml of purified T. thermophilus tRNA

  • RNase inhibitor

• Temperature considerations:

  • While T. thermophilus grows optimally at high temperatures, reconstituted systems can function at temperatures ranging from 37°C to 65°C

  • The synthesis of active proteins can occur even at 37°C, a temperature well below the minimal growth temperature for T. thermophilus

• Energy regeneration system:

  • Include enzymes for regeneration of GTP and ATP, which are necessary for sustained translation activity

  • Ensure these energy regeneration enzymes are also thermostable

What are the advantages and limitations of using T. thermophilus as a source of recombinant thermostable proteins compared to other thermophilic microorganisms?

Advantages:

• Higher expression yields in native host: T. thermophilus has emerged as a suitable host for overproducing thermozymes, with studies showing that homologous expression can yield up to 10 times more soluble and active enzyme (5 mg/L) compared to expression in E. coli (0.5 mg/L) .

• Extreme thermostability: Enzymes from T. thermophilus, such as α-galactosidase (TtGalA), demonstrate optimal activity at temperatures as high as 90°C and retain more than 40% activity over a broad pH range (5-8) .

• Valuable structural information: Extensive structural data is available for T. thermophilus translation components, facilitating structure-function studies .

• Reduced proteases and nucleases: Reconstituted thermostable cell-free protein synthesis systems from T. thermophilus contain significantly reduced nucleases and proteases, enabling in vitro engineering of proteins with improved thermostability .

Limitations:

• Expression challenges in mesophilic hosts: Proteins from T. thermophilus often do not fold properly when expressed in mesophilic hosts like E. coli, showing differences in secondary structure with fewer helices and more coils .

• Limited genetic tools: Despite recent advances, genetic manipulation tools for T. thermophilus are still less developed compared to model organisms like E. coli .

• Specific adaptation requirements: Some T. thermophilus proteins require specific conditions (like polyamines) for optimal function even at lower temperatures .

What strategies can be employed to engineer T. thermophilus EF-Ts for enhanced functionality in biotechnological applications?

Several engineering strategies can enhance T. thermophilus EF-Ts functionality:

• Targeted mutagenesis of the dimerization interface:

  • Using the crystal structure of the dimerization domain (refined to 1.7 Å resolution) , researchers can introduce mutations that either strengthen or modify the dimer interface

  • Focus on the hydrophobic core formed by Leu73, Cys190, and Phe192 residues

  • Mutations must be carefully designed to avoid disrupting the β-sandwich structure essential for dimer formation

• Homologous expression system optimization:

  • Develop improved T. thermophilus expression systems using counterselective agents like p-chlorophenylalanine for sequential introduction of mutations without antibiotic resistance markers

  • This approach allows precise genome engineering to optimize EF-Ts expression

• Hybrid systems combining thermophilic and mesophilic components:

  • Research has demonstrated functional compatibility between key translation components from T. thermophilus and E. coli

  • Engineer chimeric EF-Ts proteins incorporating beneficial properties from different species

• Disulfide bridge engineering:

  • While the physiological role of the Cys190 disulfide bridge remains unclear , strategic introduction or modification of disulfide bonds could enhance stability

  • The modification of Cys190 by iodoacetamide affects neither dimerization nor nucleotide exchange activity , suggesting tolerance for modifications at this position

• Rational design based on quaternary complex structures:

  • Modify the interface between EF-Ts and EF-Tu to enhance the formation of quaternary complexes (EF-Tu·EF-Ts)₂ or ternary complexes EF-Tu·EF-Ts₂

  • These modifications could improve the efficiency of nucleotide exchange activity