Recombinant Geobacillus thermodenitrificans Elongation factor Ts (tsf)

What is Elongation factor Ts (tsf) and what is its role in protein synthesis?

Elongation factor Ts (EF-Ts) is a guanine nucleotide exchange factor that plays a critical role in the elongation phase of protein translation. It functions by catalyzing the regeneration of active EF-Tu·GTP complex from inactive EF-Tu·GDP, allowing EF-Tu to participate in multiple rounds of aminoacyl-tRNA delivery to the ribosome . In G. thermodenitrificans, EF-Ts maintains this essential function while exhibiting remarkable thermostability, making it valuable for research in extreme environments .

What are the basic structural characteristics of G. thermodenitrificans EF-Ts?

G. thermodenitrificans EF-Ts is a protein comprising 294 amino acids with a full sequence that includes characteristic domains for nucleotide exchange activity . The recombinant protein has a UniProt accession number A4IMC4. Unlike EF-Ts from E. coli (282 aa), G. thermodenitrificans EF-Ts shares greater similarity with thermophilic organisms' translation factors. When compared to another thermophilic bacteria, Thermus thermophilus, both show adaptation to high temperatures through specific structural modifications .

What are the optimal storage conditions for recombinant G. thermodenitrificans EF-Ts?

For short-term storage (up to one week), recombinant G. thermodenitrificans EF-Ts can be stored at 4°C. For extended preservation, storing at -20°C is recommended, while -80°C provides optimal long-term stability . The shelf life in liquid form is approximately 6 months at -20°C/-80°C, while the lyophilized form remains stable for up to 12 months. It is advisable to add glycerol (5-50% final concentration, with 50% being standard) before aliquoting and freezing to prevent protein degradation during freeze-thaw cycles .

What expression systems are most effective for producing recombinant G. thermodenitrificans EF-Ts?

E. coli is the most widely used expression system for recombinant G. thermodenitrificans EF-Ts production due to its established protocols and high yield potential . When expressing thermostable proteins in mesophilic hosts like E. coli, thermal separation techniques can be employed for initial purification, as thermophilic proteins remain soluble at temperatures that denature most E. coli proteins. This advantage was demonstrated with T. thermophilus EF-Ts, which could be readily separated from E. coli proteins using heat treatment . For optimal expression, codon optimization may be necessary to account for codon usage differences between G. thermodenitrificans and E. coli .

What purification strategy offers the highest purity for recombinant G. thermodenitrificans EF-Ts?

A multi-step purification strategy is recommended for obtaining highly pure recombinant G. thermodenitrificans EF-Ts:

• Initial thermal separation (heating lysate to 55-60°C for 10 minutes) to denature E. coli proteins

• Affinity chromatography using histidine or other suitable tags

• Ion-exchange chromatography for removing remaining contaminants

• Size exclusion chromatography for final polishing

This approach typically yields protein with >85% purity as assessed by SDS-PAGE . For applications requiring higher purity, additional chromatography steps may be employed. The thermal stability of G. thermodenitrificans EF-Ts allows for heat treatment during purification without activity loss, providing an advantage over mesophilic proteins .

How can researchers confirm the functionality of purified recombinant G. thermodenitrificans EF-Ts?

Functionality of purified G. thermodenitrificans EF-Ts can be confirmed through multiple complementary approaches:

• Nucleotide exchange assay: Measuring the rate of GDP/GTP exchange on EF-Tu in the presence of purified EF-Ts

• Complex formation analysis: Using gel permeation chromatography to detect formation of the EF-Tu·EF-Ts complex

• Polyacrylamide gel electrophoresis: Analyzing the quaternary (EF-Tu·EF-Ts)₂ and ternary EF-Tu·EF-Ts₂ complexes

• Thermal stability testing: Confirming activity retention after heat treatment at 60-65°C

These methods provide comprehensive verification of both structural integrity and functional activity of the purified protein .

How does the structure of G. thermodenitrificans EF-Ts differ from mesophilic counterparts?

G. thermodenitrificans EF-Ts exhibits several structural adaptations that contribute to its thermostability compared to mesophilic counterparts:

• More compact structure with fewer flexible regions

• Higher proportion of charged residues forming ionic interactions

• Potentially increased number of hydrophobic interactions in the core

• Specific amino acid substitutions that favor stability at high temperatures

Comparative studies with EF-Ts from E. coli show that thermophilic EF-Ts typically have adaptations that increase rigidity while maintaining functional flexibility at elevated temperatures . While T. thermophilus EF-Ts is considerably shorter than E. coli EF-Ts (differing by 86 amino acids), this differential is species-specific and may not apply to G. thermodenitrificans EF-Ts .

What is known about potential post-translational modifications of G. thermodenitrificans EF-Ts?

While specific post-translational modifications of G. thermodenitrificans EF-Ts have not been extensively characterized, studies of related thermophilic EF-Ts proteins provide insights. In T. thermophilus, EF-Ts can form homodimers through disulfide bridges between cysteine residues, although the physiological relevance remains unclear as these were only detected in purified proteins and not in cell extracts .

The G. thermodenitrificans EF-Ts sequence contains cysteine residues that could potentially form similar bonds, but experimental confirmation is required. Modification of cysteine residues by agents like iodoacetamide could affect protein function, as demonstrated in T. thermophilus where Cys78 modification yielded inactive EF-Ts .

How does G. thermodenitrificans EF-Ts interact with EF-Tu during protein synthesis?

G. thermodenitrificans EF-Ts interacts with EF-Tu to catalyze the exchange of GDP for GTP through the following mechanism:

• EF-Ts binds to the EF-Tu·GDP complex

• This binding induces conformational changes that decrease EF-Tu's affinity for GDP

• GDP is released, forming a binary EF-Tu·EF-Ts complex

• GTP binds to EF-Tu, displacing EF-Ts

• The regenerated EF-Tu·GTP complex can then participate in delivering aminoacyl-tRNA to the ribosome

Research indicates that thermophilic EF-Ts/EF-Tu interactions maintain similar functional mechanisms to mesophilic systems but with enhanced stability at elevated temperatures. Both quaternary (EF-Tu·EF-Ts)₂ complexes and ternary EF-Tu·EF-Ts₂ complexes have been detected using gel permeation chromatography and polyacrylamide gel electrophoresis in thermophilic systems .

What experimental approaches are most effective for analyzing G. thermodenitrificans EF-Ts binding kinetics with EF-Tu?

Several complementary approaches provide comprehensive analysis of EF-Ts/EF-Tu binding kinetics:

For thermostable proteins like G. thermodenitrificans EF-Ts, these experiments should be conducted at physiologically relevant temperatures (45-65°C) to accurately represent in vivo conditions . Comparative studies with mesophilic EF-Ts at various temperatures can provide insights into thermoadaptation mechanisms.

How does the ribosomal binding site of EF-Ts relate to other elongation factors?

The binding of EF-Ts to ribosomes is indirectly related to other elongation factors through its interaction with EF-Tu. Research has shown that elongation factors EF-Tu and EF-G appear to interact at related sites on the ribosome . When EF-G binds to ribosomes, it inhibits the subsequent binding of the aminoacyl-tRNA·EF-Tu·GTP ternary complex, suggesting overlapping binding regions .

Although EF-Ts primarily functions by interacting with EF-Tu in solution, the recycling of EF-Tu·GDP to EF-Tu·GTP by EF-Ts is essential for maintaining the pool of active EF-Tu molecules that can participate in ribosomal binding. Understanding these spatial relationships is crucial for comprehending the complete elongation cycle in thermophilic translation systems .

What mechanisms contribute to the thermostability of G. thermodenitrificans EF-Ts?

G. thermodenitrificans EF-Ts demonstrates remarkable thermostability, consistent with the organism's growth temperature range of 45-73°C (optimum 65°C) . Several mechanisms likely contribute to this thermostability:

• Amino acid composition: Increased proportion of charged residues forming salt bridges and hydrogen bonds

• Protein compactness: Reduction in flexible loops and termini that are prone to unfolding

• Hydrophobic core packing: Optimized van der Waals interactions in the protein core

• Quaternary structure: Potential formation of stabilizing oligomeric structures similar to those observed in other thermophilic EF-Ts proteins

• Reduced conformational entropy: Stabilizing mutations that decrease the entropy of the unfolded state

Interestingly, in Bacillus stearothermophilus (a closely related thermophile), the heat stabilities of EF-Tu and EF-Ts are reversed with respect to E. coli factors, with EF-Tu being the more stable protein . This pattern may also apply to G. thermodenitrificans and represents an important adaptation in thermophilic translation systems.

How can researchers experimentally assess the thermostability of recombinant G. thermodenitrificans EF-Ts?

Several complementary methods can be employed to assess thermostability:

• Thermal inactivation assays: Measuring residual activity after incubation at increasing temperatures

• Differential Scanning Calorimetry (DSC): Determining the melting temperature (Tm) and enthalpy of unfolding

• Circular Dichroism (CD) spectroscopy: Monitoring secondary structure changes during thermal denaturation

• Intrinsic fluorescence spectroscopy: Tracking tertiary structure changes with increasing temperature

• Limited proteolysis: Comparing susceptibility to proteolytic degradation at various temperatures

For G. thermodenitrificans EF-Ts, these assays should be performed across a wide temperature range (30-90°C) to fully characterize its thermal stability profile and identify the temperature at which 50% activity is lost (T1/2) . Comparative analysis with EF-Ts from mesophilic organisms provides valuable insights into thermoadaptation mechanisms.

How do environmental factors beyond temperature affect G. thermodenitrificans EF-Ts stability and function?

The stability and function of G. thermodenitrificans EF-Ts are influenced by various environmental factors:

• pH: While optimal pH has not been specifically reported for G. thermodenitrificans EF-Ts, related thermophilic proteins typically show highest stability and activity at neutral to slightly alkaline pH

• Ionic strength: Higher salt concentrations may enhance thermostability by strengthening electrostatic interactions

• Divalent cations: Mg²⁺ ions are crucial for nucleotide binding and exchange functions

• Reducing/oxidizing conditions: Potential disulfide bonds may form under oxidizing conditions, potentially affecting oligomerization and activity

• Organic solvents: Information from related thermophilic enzymes suggests G. thermodenitrificans EF-Ts may maintain activity in the presence of certain organic solvents, making it potentially useful in non-conventional reaction conditions

Understanding these factors is essential for optimizing experimental conditions when working with this protein both in vitro and in heterologous expression systems.

How does G. thermodenitrificans EF-Ts compare to EF-Ts from other thermophilic bacteria?

G. thermodenitrificans EF-Ts shares characteristics with other thermophilic EF-Ts proteins while maintaining species-specific features:

These differences reflect evolutionary adaptations to specific ecological niches while maintaining the core function of guanine nucleotide exchange during protein synthesis .

What methodological approaches are most effective for comparing the functional differences between thermophilic and mesophilic EF-Ts proteins?

Effective comparative analysis between thermophilic and mesophilic EF-Ts requires multi-faceted approaches:

• Sequence alignment and structural analysis: Identifying conserved domains and variable regions that might contribute to thermostability

• Homology modeling: Predicting structural differences in the absence of crystal structures

• Chimeric protein construction: Creating fusion proteins between thermophilic and mesophilic EF-Ts domains to identify thermostability determinants

• Parallel activity assays: Conducting nucleotide exchange assays at various temperatures for both proteins

• Mutagenesis studies: Introducing point mutations to convert thermophilic to mesophilic characteristics and vice versa

These approaches have successfully identified that elongation factors from thermophiles like G. thermodenitrificans have evolved specific adaptations for function at elevated temperatures while maintaining essential interactions with their translation partners .

How is the tsf gene organized in the G. thermodenitrificans genome?

The tsf gene encoding Elongation factor Ts in G. thermodenitrificans is part of the complete 3,550,319-bp chromosome . While the specific genetic organization of tsf in G. thermodenitrificans hasn't been described in detail in the provided search results, insights can be drawn from related organisms. In E. coli, the tsf gene is located near dapD at approximately 4 minutes on the genetic map, and is distinct from the chromosomal locations where many ribosomal protein genes and other elongation factor genes (fus, tufA, and tufB) are clustered .

Comparative genomic analysis would be needed to determine if G. thermodenitrificans maintains a similar genetic organization or if the extreme thermophilic adaptation has led to different genomic arrangements. The G. thermodenitrificans genome contains 3,499 predicted ORFs and 11 rRNA operons, with genes covering 86% of the genome .

What approaches are most effective for cloning and expressing the G. thermodenitrificans tsf gene in heterologous hosts?

Effective strategies for cloning and expressing G. thermodenitrificans tsf gene include:

• Codon optimization: Adjusting codon usage to match the host organism for improved expression

• Vector selection: Using expression vectors with strong, inducible promoters (T7, tac) for controlled expression

• Host strain selection: Choosing E. coli strains designed for expression of thermophilic proteins

• Growth conditions optimization: Lower induction temperatures (25-30°C) often improve folding of thermostable proteins in mesophilic hosts

• Fusion tags: Addition of solubility-enhancing tags (MBP, SUMO) can improve expression yields

For transformation of thermophilic hosts, specialized electroporation protocols may be required. For example, P. thermoglucosidasius (a related thermophile) requires specific electroporation parameters: 2.5 kV, 10 μF, 600 Ω, followed by recovery at 52°C .

How do regulatory elements controlling tsf expression differ between thermophilic and mesophilic bacteria?

Regulatory elements controlling gene expression in thermophiles like G. thermodenitrificans must function at elevated temperatures, necessitating specific adaptations:

• Promoter design: Thermophilic promoters typically have higher GC content in critical regions to maintain DNA duplex stability at elevated temperatures

• Ribosome binding sites: Stronger Shine-Dalgarno sequences may be required to ensure stable mRNA-ribosome interactions at high temperatures

• mRNA stability: Secondary structures in mRNAs from thermophiles are typically more stable to prevent premature degradation

• Transcription factors: Regulatory proteins controlling expression must themselves be thermostable

Research in G. thermodenitrificans and related thermophiles has identified specific promoter collections that function reliably at elevated temperatures, which is essential for expressing genes like tsf in their native context . When designing expression systems for tsf in heterologous hosts, these thermophilic regulatory elements should be considered if expression at elevated temperatures is desired .

How can recombinant G. thermodenitrificans EF-Ts be utilized in cell-free protein synthesis systems?

Recombinant G. thermodenitrificans EF-Ts offers several advantages for thermostable cell-free protein synthesis (CFPS) systems:

• High-temperature CFPS: Enabling protein synthesis at 50-65°C, which can improve folding of thermostable target proteins

• Extended reaction lifetimes: Thermostable translation factors like EF-Ts maintain activity longer than mesophilic counterparts

• Reduced contamination risk: Higher temperature operation minimizes microbial contamination during extended CFPS reactions

• Compatibility with difficult proteins: Some proteins prone to misfolding or aggregation at lower temperatures may fold properly at elevated temperatures

Implementation requires careful optimization of all components, including:

• Balanced ratios of EF-Ts to EF-Tu for optimal recycling

• Compatible ribosomes (either from thermophiles or engineered for thermostability)

• Heat-stable energy regeneration systems

• Temperature-optimized buffer conditions

These high-temperature CFPS systems are particularly valuable for producing thermostable enzymes for industrial applications.

What experimental protocols should be followed when using G. thermodenitrificans EF-Ts as a thermostable component in molecular biology applications?

When utilizing G. thermodenitrificans EF-Ts in molecular biology applications, the following protocol considerations are important:

• Reconstitution: Protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL with 5-50% glycerol for stability

• Buffer compatibility: Optimal activity typically requires:

  • pH 7.0-8.0 buffer systems stable at high temperatures (HEPES, phosphate)

  • 5-10 mM Mg²⁺ for nucleotide exchange function

  • 50-150 mM monovalent cations (K⁺ or NH₄⁺)

• Temperature parameters: Reactions should be performed at 45-65°C for optimal activity, with experimental controls to verify function

• Storage during experiments: Keep on ice when not in use and avoid repeated freeze-thaw cycles

• Quality control: Verify activity before use through nucleotide exchange assays with compatible EF-Tu

For advanced applications like reconstituted translation systems, it's essential to ensure compatibility between all components, including G. thermodenitrificans EF-Ts, EF-Tu, and other translation factors .

What are the most promising research directions for further characterizing G. thermodenitrificans EF-Ts structure-function relationships?

Several promising research directions would advance our understanding of G. thermodenitrificans EF-Ts:

• Structural determination: X-ray crystallography or cryo-electron microscopy of free EF-Ts and the EF-Tu·EF-Ts complex to elucidate thermostability mechanisms

• Molecular dynamics simulations: Computational analysis of protein movements at different temperatures to identify flexible regions and stability-determining interactions

• Directed evolution: Creating enhanced variants with even greater thermostability or modified functionality

• Domain swapping experiments: Exchanging domains between thermophilic and mesophilic EF-Ts to pinpoint thermostability determinants

• In vivo studies: Investigating the physiological role of potential EF-Ts dimerization in G. thermodenitrificans under various growth conditions

• Biophysical characterization: Detailed analysis of folding/unfolding pathways using techniques like hydrogen-deuterium exchange mass spectrometry

These approaches would provide insights not only into G. thermodenitrificans EF-Ts but also into general principles of protein thermostability applicable to protein engineering and synthetic biology applications .