Recombinant Thermotoga neapolitana Elongation factor Tu (tuf)

Basic Research Questions

• What is Thermotoga neapolitana Elongation factor Tu (tuf) and what is its role in protein translation?

  Elongation factor Tu (EF-Tu), encoded by the tuf gene, is a critical GTP-binding protein in the translation elongation phase of protein synthesis. During translation, EF-Tu forms a ternary complex with GTP and aminoacyl-tRNA (aa-tRNA), delivering the aa-tRNA to the A-site of the ribosome. Following GTP hydrolysis, EF-Tu-GDP is released from the ribosome as an inactive form. Subsequently, elongation factor Ts (EF-Ts) binds to EF-Tu, facilitating the exchange of GDP with GTP, regenerating the active EF-Tu-GTP complex for delivering another aa-tRNA to begin a new translation cycle . This mechanism ensures accurate and efficient protein synthesis by properly mediating codon-anticodon recognition during the elongation process.

• Why is Thermotoga neapolitana's EF-Tu of particular interest to researchers?

  Thermotoga neapolitana is a hyperthermophilic bacterium that grows optimally at temperatures between 77°C and 85°C . Its EF-Tu is of significant interest to researchers for several reasons:

  • It maintains functionality at extremely high temperatures, making it an excellent model for studying protein thermostability mechanisms

  • Understanding the structural adaptations that enable functionality at high temperatures provides insights for protein engineering applications

  • Comparing hyperthermophilic EF-Tu with mesophilic homologs reveals evolutionary strategies for adaptation to extreme environments

  • T. neapolitana represents an ancient evolutionary lineage, providing potential insights into the evolution of the translation apparatus

  • Its thermostable properties make it potentially valuable for high-temperature biochemical applications and in vitro translation systems

• What are the optimal storage and handling conditions for recombinant Thermotoga neapolitana EF-Tu?

  Based on recommended protocols for similar recombinant proteins from T. neapolitana:

  • Liquid formulations should be stored at -20°C/-80°C with an expected shelf life of approximately 6 months

  • Lyophilized formulations can be stored at -20°C/-80°C with a longer shelf life of approximately 12 months

  • Repeated freezing and thawing cycles should be avoided to maintain protein integrity

  • Working aliquots can be stored at 4°C for up to one week

  • A storage buffer containing Tris-based buffer with 50% glycerol is typically used to maintain stability

  • For reconstitution of lyophilized protein, gentle mixing rather than vortexing is recommended

  • Proper storage in single-use aliquots can significantly extend the functional lifetime of the protein

• How does the structure of EF-Tu contribute to its function in protein translation?

  While the search results don't provide the specific structure of T. neapolitana EF-Tu, typical bacterial EF-Tu proteins have a three-domain structure (domains I, II, and III) with distinct functional characteristics:

  • Domain I contains the GTP/GDP binding site and undergoes significant conformational changes upon nucleotide exchange

  • Structural domains I and III are typically closer together than domains I and II, allowing domains I and III to interact face-to-face through side-chain interactions

  • The three domains form a flat triangular arrangement that ensures a high degree of interdomain flexibility, facilitating the binding of EF-Tu to various substrates during peptide synthesis

  • In thermophilic organisms, additional stabilizing features like ion pairs and hydrophobic interactions likely contribute to maintaining functional structure at high temperatures

• What is the relationship between EF-Tu and EF-Ts in T. neapolitana?

  The interaction between EF-Tu and EF-Ts is crucial for translation elongation:

  • EF-Ts functions as a guanine nucleotide exchange factor for EF-Tu, facilitating the exchange of GDP for GTP after each round of aa-tRNA delivery

  • T. neapolitana possesses a Mitochondrial Translation Elongation Factor EF-Ts Tsf1 protein of 197 amino acids that interacts with EF-Tu

  • While EF-Tu is highly conserved across species, EF-Ts shows more variation between different species

  • The proportion of EF-Ts that binds and reactivates with EF-Tu differs between bacterial species

  • This interaction is essential for recycling EF-Tu into its active GTP-bound form to continue the translation elongation cycle

Advanced Research Questions

• What structural features might explain the thermostability of Thermotoga neapolitana EF-Tu?

  Thermophilic proteins like T. neapolitana EF-Tu typically employ several adaptations for thermostability:

  • Ion Pair Networks: Extended networks of salt bridges, particularly at domain interfaces, that provide electrostatic stability at high temperatures

  • Strategic Proline Positioning: Prolines often appear in position 2 of solvent-exposed β-turns, in coils within loops, or at the N-cap of α-helices to restrict conformational flexibility at high temperatures

  • Surface Charge Optimization: Higher density of charged residues (particularly Arg and Glu) on the protein surface that form stabilizing salt bridges

  • Loop Modifications: Shorter loop regions with restricted flexibility compared to mesophilic homologs

  • Hydrophobic Core Packing: Tighter packing of hydrophobic residues in the protein core

  Methodological approach for investigation:

  • Comparative sequence and structural analysis with mesophilic homologs

  • Site-directed mutagenesis to verify the contribution of specific stabilizing features

  • Thermal denaturation studies to quantify stability changes from specific mutations

  • Molecular dynamics simulations at different temperatures to identify key stabilizing interactions

• How can researchers optimize heterologous expression systems for T. neapolitana EF-Tu?

  Optimal expression of thermophilic proteins in mesophilic hosts requires careful consideration of:

  Parameter	Optimization Strategy
  Expression Host	E. coli BL21(DE3) strains; Rosetta strains if rare codons are present
  Vector System	pET vectors with T7 promoter for controlled induction
  Induction Temperature	15-25°C (lower than standard) to improve solubility
  Induction Duration	Extended expression times (16-24 hours)
  Solubility Enhancement	Fusion with solubility tags (MBP, SUMO, Thioredoxin); co-expression with chaperones
  Post-extraction	Heat treatment (50-60°C) to remove host proteins while retaining thermostable target
  Purification Buffer	Addition of stabilizers (glycerol, specific salts)

  The purification process should include:

  • Initial heat treatment step to leverage the thermostability of T. neapolitana EF-Tu

  • Affinity chromatography using a suitable tag (e.g., His-tag)

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography for final polishing and buffer exchange

  • Quality assessment by SDS-PAGE to confirm purity greater than 85%

• What biophysical techniques are most valuable for characterizing the thermodynamic properties of T. neapolitana EF-Tu?

  A comprehensive biophysical characterization would include:

  • Differential Scanning Calorimetry (DSC): Determines melting temperature (Tm), enthalpy (ΔH), and heat capacity changes (ΔCp)

  • Circular Dichroism (CD): Monitors secondary structure content and thermal unfolding transitions at different temperatures

  • Isothermal Titration Calorimetry (ITC): Quantifies binding thermodynamics with ligands (GTP/GDP) and interaction partners (EF-Ts, aa-tRNA)

  • Fluorescence Spectroscopy: Tracks conformational changes through intrinsic tryptophan fluorescence or external probes

  • Dynamic Light Scattering (DLS): Assesses size distribution and aggregation propensity at different temperatures

  Experimental design should include:

  • Temperature ranges from ambient to 85°C (or higher) to capture the full stability profile

  • Comparison with mesophilic homologs under identical conditions

  • Multiple buffer conditions to assess the role of salts and pH on thermostability

  • Nucleotide-bound and nucleotide-free states to understand the impact of ligand binding on stability

• How does the interaction between T. neapolitana EF-Tu and EF-Ts differ from other bacterial species?

  From the search results, we know that:

  • EF-Ts proteins show less conservation between species than EF-Tu proteins

  • The proportion of EF-Ts that binds and reactivates with EF-Tu differs between bacterial species

  • T. neapolitana EF-Ts has a sequence of 197 amino acids as shown in search result

  • In some thermophilic bacteria like Thermus thermophilus, EF-Ts functions as a dimer for nucleotide exchange with EF-Tu

  Methodological approaches to characterize these interactions include:

  • Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI) to measure binding kinetics at different temperatures

  • Isothermal Titration Calorimetry (ITC) to determine thermodynamic parameters of the interaction

  • Nucleotide exchange assays to quantify the functional efficiency of the EF-Tu/EF-Ts interaction

  • Pull-down assays using tagged proteins to identify potential additional interaction partners

  • Crystallographic or cryo-EM studies of the EF-Tu/EF-Ts complex to map the interaction interface

• How can recombinant T. neapolitana EF-Tu be utilized in in vitro translation systems?

  Thermostable translation factors offer unique advantages for in vitro translation systems:

  • High-Temperature Translation: Development of thermostable cell-free protein synthesis systems that can operate at elevated temperatures (50-70°C)

  • Enhanced System Stability: Longer reaction lifetimes compared to mesophilic components

  • Difficult Template Translation: More efficient translation of structured mRNAs at higher temperatures where secondary structures are destabilized

  Implementation methodology:

  Component	Consideration
  EF-Tu Preparation	Ensure high purity (>90%) and confirm activity through GTPase assays
  Buffer Composition	Optimize for high-temperature activity (salt concentration, pH stability)
  Additional Components	Purify or source other thermostable translation components (ribosomes, EF-G, initiation factors)
  Compatibility Testing	Assess functionality with mesophilic components if developing hybrid systems
  Performance Metrics	Measure translation rates, protein yield, and translation fidelity at different temperatures

  Applications include production of thermostable proteins that may fold more effectively at higher temperatures and development of more robust cell-free protein synthesis platforms.

• What methods are most effective for studying conformational changes in T. neapolitana EF-Tu during the GTP/GDP cycle?

  EF-Tu undergoes significant conformational changes between GTP and GDP-bound states that are critical to its function:

  • X-ray Crystallography: Obtain high-resolution structures in different nucleotide-bound states using non-hydrolyzable GTP analogs

  • Cryo-Electron Microscopy: Visualize EF-Tu in complex with ribosomes to capture functional states

  • FRET (Förster Resonance Energy Transfer): Monitor real-time conformational changes by introducing fluorescent labels at strategic positions

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map protein dynamics and solvent accessibility changes in different nucleotide states

  • NMR Spectroscopy: Characterize local conformational changes, though challenging for a protein of this size

  Experimental design should include:

  • Comparison of conformational changes at different temperatures (25°C vs. 70-85°C)

  • Analysis of the effect of EF-Ts on conformational dynamics

  • Investigation of how thermostability mechanisms might influence the conformational cycle

  • Time-resolved studies to capture transient intermediates in the nucleotide exchange process

• What are the challenges in crystallizing T. neapolitana EF-Tu for structural studies, and how can they be addressed?

  Crystallizing thermophilic proteins presents specific challenges:

  • Conformational Heterogeneity: EF-Tu exists in different conformational states depending on bound nucleotide

  • Nucleotide State Control: Ensuring homogeneous nucleotide-binding state is critical

  • Complex Formation Considerations: EF-Tu may need to be crystallized in complex with binding partners

  Methodological approaches to overcome these challenges:

  Challenge	Solution Strategy
  Conformational Heterogeneity	Use of non-hydrolyzable GTP analogs (GMPPNP, GMPPCP) or GDP to lock specific conformations
  Crystallization Conditions	Sparse matrix screening at different temperatures (4°C, room temperature, elevated temperatures)
  Protein Engineering	Surface entropy reduction mutations to promote crystal contacts
  Complex Stabilization	Co-crystallization with EF-Ts or aa-tRNA to stabilize specific conformations
  Alternative Approaches	Cryo-electron microscopy if crystallization proves challenging

  Screening strategies should include specialized conditions for thermophilic proteins, including higher salt concentrations and additives that enhance thermostability during the crystallization process.

• How can researchers investigate the role of specific amino acid residues in the thermostability of T. neapolitana EF-Tu?

  Site-directed mutagenesis combined with functional and structural analysis is the primary approach:

  • Identify potential stabilizing residues through sequence alignment with mesophilic homologs

  • Target ion pairs, particularly those forming networks that may contribute to thermostability

  • Investigate proline residues in β-turns, coils within loops, or at the N-cap of α-helices that may restrict conformational flexibility

  • Examine the role of charged residues (especially Arg-Glu pairs) that form salt bridges

  Experimental workflow:

  1. Generate single and multiple point mutations in recombinant T. neapolitana EF-Tu

  2. Express and purify mutant proteins using optimized protocols

  3. Assess thermostability changes through thermal denaturation assays (CD, DSC)

  4. Measure functional properties (GTPase activity, nucleotide exchange rates)

  5. Determine structure-function relationships by correlating stability changes with specific mutations

  For example, mutations similar to the Arg241Ala mutation in T. maritima indoleglycerol phosphate synthase, which increased enzyme denaturation rate by a factor of almost 3 at 85.5°C , could be introduced at analogous positions in T. neapolitana EF-Tu to test the contribution of specific ion pairs to thermostability.