Recombinant Chloroflexus aurantiacus Elongation factor Tu (tuf1)

What are the optimal storage and handling conditions for Recombinant Chloroflexus aurantiacus Elongation factor Tu?

For optimal stability and activity retention of Recombinant C. aurantiacus Elongation factor Tu, researchers should follow these methodological guidelines:

Storage Recommendations:

• Short-term storage (up to one week): 4°C

• Standard storage: -20°C

• Extended storage: -20°C to -80°C

Handling Protocol:

• When receiving lyophilized protein, briefly centrifuge the vial before opening to collect all material at the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (with 50% being standard practice)

• Prepare small working aliquots to minimize freeze-thaw cycles

• Store aliquots at recommended temperatures

The shelf life varies depending on formulation:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: approximately 12 months at -20°C/-80°C

IMPORTANT: Repeated freeze-thaw cycles significantly reduce protein stability and activity. Researchers should track the number of freeze-thaw cycles each aliquot undergoes and discard samples after 3-5 cycles for most reliable results .

How is Chloroflexus aurantiacus positioned evolutionarily among photosynthetic organisms?

Chloroflexus aurantiacus occupies a fascinating position in the evolutionary history of photosynthetic organisms. As a member of the Chloroflexi phylum, it represents one of the earliest diverging photosynthetic bacterial lineages.

The unique features that distinguish C. aurantiacus in evolutionary context include:

• Photosynthetic apparatus characteristics: Contains a type II reaction center similar to purple bacteria, but with distinctive modifications. The M-subunit of the reaction center consists of 306 amino acid residues with a blocked N-terminus and replacement of a histidine (that normally coordinates magnesium of an accessory bacteriochlorophyll in purple bacteria) with leucine .

• Novel electron transfer mechanisms: Possesses a unique multi-subunit membrane-bound electron transfer complex containing seven subunits, two of which are c-type cytochromes. This complex functionally replaces the cytochrome bc or bf complex found in many other photosynthetic organisms .

• Metabolic versatility: Capable of photoheterotrophic, photoautotrophic, and chemoheterotrophic growth, suggesting an adaptable metabolism that may reflect early evolutionary strategies.

These characteristics make C. aurantiacus an excellent model organism for studying the evolution of photosynthesis and electron transfer mechanisms. The distinct features of its proteins, including Elongation factor Tu, may reflect adaptations to its thermophilic lifestyle and unique evolutionary position .

What expression systems are recommended for producing Recombinant Chloroflexus aurantiacus Elongation factor Tu?

Based on current research protocols, the recommended expression system for producing Recombinant C. aurantiacus Elongation factor Tu is a yeast-based system. This methodological approach offers several advantages for this specific protein:

• Expression System Details:

  • Host organism: Yeast (specific strain optimization may be required)

  • Target protein: Full-length (1-401 amino acids) Elongation factor Tu

  • Expected purity: >85% by SDS-PAGE analysis

  • Product identity: Verified by mass spectrometry or Western blotting

• Methodological Workflow:

  • Gene synthesis or amplification from C. aurantiacus genomic DNA

  • Cloning into appropriate yeast expression vector

  • Transformation and selection of high-expressing clones

  • Optimization of induction conditions

  • Purification via affinity chromatography (tag-specific)

  • Quality control testing

While yeast is the documented expression system, researchers may consider alternative systems based on specific experimental requirements:

The choice of expression system should be guided by the intended application, with yeast being preferred when native-like protein conformation and potential post-translational modifications are important .

What protocols are recommended for assessing the GTPase activity of Recombinant Chloroflexus aurantiacus Elongation factor Tu?

The assessment of GTPase activity is crucial for functional characterization of Recombinant C. aurantiacus Elongation factor Tu. A comprehensive methodological approach should include multiple complementary assays:

• Malachite Green Phosphate Assay Protocol:

  • Prepare reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 100 mM KCl)

  • Combine 1-5 μM purified EF-Tu with 50-200 μM GTP

  • Incubate at both standard (37°C) and thermophilic (55°C) conditions

  • At timed intervals, withdraw aliquots and terminate reactions with malachite green reagent

  • Measure absorbance at 650 nm and calculate released phosphate against standards

• HPLC Nucleotide Analysis:

  • Set up reactions as above

  • Quench with EDTA at predetermined timepoints

  • Analyze GTP/GDP ratios by reversed-phase HPLC

  • Integrate peak areas to determine reaction progression

• Temperature and pH Optimization:
  For thermophilic EF-Tu from C. aurantiacus, systematic optimization is essential:

  Temperature (°C)	Relative Activity (%)	pH	Relative Activity (%)	[Mg²⁺] (mM)	Relative Activity (%)
  30	20-30	6.5	50-60	5	60-70
  40	50-60	7.0	80-90	10	90-100
  50	80-90	7.5	95-100	15	80-90
  60	90-100	8.0	70-80	20	70-80
  70	60-70	8.5	40-50	25	50-60

• Real-time Kinetics via Fluorescence:

  • Employ fluorescent GTP analogs (mant-GTP)

  • Monitor binding and hydrolysis through fluorescence changes

  • Determine kon and koff rates for nucleotide interactions

• Control Experiments:

  • Negative control: Heat-denatured EF-Tu

  • Positive control: E. coli EF-Tu with established kinetic parameters

  • Specificity control: ATP versus GTP substrate preference

This methodological framework allows researchers to comprehensively characterize the GTPase activity of C. aurantiacus EF-Tu and compare it with homologs from other bacterial species, particularly with respect to thermostability and temperature optima.

How does the thermostability of Chloroflexus aurantiacus Elongation factor Tu compare to mesophilic homologs, and what structural features contribute to these differences?

As a protein from a thermophilic organism, C. aurantiacus Elongation factor Tu exhibits distinctive thermostability properties compared to mesophilic homologs. A systematic investigation reveals both the extent and mechanisms of this enhanced stability:

• Comparative Thermal Stability Assessment:
  Experimental approaches for quantitative comparison include:

  • Differential scanning calorimetry (DSC) to determine melting temperatures (Tm)

  • Circular dichroism (CD) spectroscopy to monitor secondary structure loss

  • Activity retention assays following thermal challenge

  Organism	Growth Temperature	EF-Tu Tm Value	Activity Half-life at 60°C
  C. aurantiacus	50-60°C	~75-85°C (estimated)	Hours
  E. coli	37°C	~45-55°C	Minutes
  T. thermophilus	65-70°C	~80-90°C	Days

• Structural Features Contributing to Thermostability:
  Analysis of the amino acid sequence reveals several thermoadaptive features:

  • Electrostatic Interactions: Increased proportion of charged residues (Arg, Lys, Glu, Asp) forming stabilizing salt bridges

  • Hydrophobic Core: Enhanced hydrophobic packing through higher proportion of branched amino acids

  • Loop Stabilization: Strategic proline residues in loop regions reducing conformational entropy

  • Thermolabile Residue Reduction: Lower content of asparagine and glutamine that are prone to deamidation at high temperatures

  • Surface Features: Potential reduction in surface loop length and increased surface hydrophilicity

• Methodological Approaches for Structure-Stability Relationships:

  • Homology modeling based on known EF-Tu structures

  • Molecular dynamics simulations at various temperatures

  • Site-directed mutagenesis targeting putative stabilizing residues

  • X-ray crystallography or cryo-EM to determine precise structural features

Understanding these thermostability mechanisms has significant implications beyond basic science, including potential applications in:

• Protein engineering for enhanced thermostability

• Development of heat-resistant translation systems for biotechnology

• Insight into evolutionary strategies for adaptation to extreme environments

What is the role of Elongation factor Tu in the context of Chloroflexus aurantiacus' unique photosynthetic and electron transfer systems?

The intersection between Elongation factor Tu function and C. aurantiacus' distinctive photosynthetic apparatus presents an intriguing research area. While EF-Tu's primary role in translation is conserved, its activity may be specifically adapted to support the organism's unique energy metabolism:

• Integration with Photosynthetic Machinery:
  C. aurantiacus possesses a photosynthetic reaction center with distinctive features compared to purple bacteria, including a modified M-subunit with a blocked N-terminus and replacement of a conserved histidine with leucine . EF-Tu's role in this context includes:

  • Translation of photosynthetic proteins with appropriate efficiency and accuracy

  • Potential coordinated regulation with light-harvesting systems

  • Adaptation to expression needs across different growth modes (photoheterotrophic vs. chemoheterotrophic)

• Connection to Novel Electron Transfer Complexes:
  C. aurantiacus contains a unique seven-subunit membrane-bound electron transfer complex that functionally replaces the cytochrome bc or bf complex found in other photosynthetic organisms . This suggests:

  • Requirements for specialized translation of these novel components

  • Potential co-evolution of translation machinery with electron transfer innovations

  • Adaptation to the energy demands of protein synthesis in a thermophilic phototroph

• Methodological Approaches to Investigate These Relationships:

  • Comparative proteomics under different growth conditions to correlate EF-Tu abundance with photosynthetic components

  • Ribosome profiling to assess translation efficiency of photosynthesis-related mRNAs

  • Analysis of tuf1 gene expression regulation in response to light and redox state

  • Co-immunoprecipitation studies to identify potential regulatory interactions

This integration of fundamental translation processes with specialized energy metabolism represents an important aspect of C. aurantiacus' evolutionary adaptation to its ecological niche, combining thermophilic lifestyle with photosynthetic capabilities.

What approaches can be used to investigate potential post-translational modifications of Chloroflexus aurantiacus Elongation factor Tu?

Post-translational modifications (PTMs) can significantly impact protein function, and their investigation in C. aurantiacus EF-Tu requires a multi-faceted methodological approach:

• Mass Spectrometry-Based PTM Mapping:

  • Sample preparation: Purify recombinant protein to >95% homogeneity

  • Enzymatic digestion: Multiple proteases (trypsin, chymotrypsin, Glu-C) for comprehensive sequence coverage

  • LC-MS/MS analysis: High-resolution instruments with various fragmentation methods (HCD, ETD)

  • Bioinformatic analysis: Search algorithms allowing for variable modifications

• PTM-Specific Enrichment Strategies:

  PTM Type	Enrichment Method	Detection Approach	Control
  Phosphorylation	TiO₂ or IMAC	Neutral loss scanning	Lambda phosphatase treatment
  Methylation	Antibody-based	Diagnostic fragment ions	Synthetic methylated peptides
  Acetylation	Anti-acetyl lysine antibodies	Diagnostic mass shift	HDAC treatment
  Glycosylation	Lectin affinity	Glycosidase treatment	PNGase F digestion

• Functional Impact Assessment:

  • Site-directed mutagenesis of modified residues

  • Comparative activity assays (GTP hydrolysis, tRNA binding)

  • Structural studies comparing modified and unmodified forms

  • In vivo studies correlating modification state with growth conditions

• Context-Specific Analysis:

  • Compare PTM profiles under different growth conditions:

    • Different temperatures (30°C vs. 55°C)

    • Light vs. dark growth

    • Aerobic vs. anaerobic conditions

  • Quantitative PTM analysis using stable isotope labeling

  • PTM crosstalk analysis for multiple modifications

This comprehensive approach would provide insights into how PTMs might modulate EF-Tu function in response to environmental conditions, potentially contributing to C. aurantiacus' adaptation to its thermophilic photosynthetic lifestyle.

What structural biology techniques are most appropriate for studying Recombinant Chloroflexus aurantiacus Elongation factor Tu?

Elucidating the three-dimensional structure of C. aurantiacus EF-Tu requires careful selection and optimization of structural biology techniques. A comprehensive methodological framework includes:

This multi-technique approach allows researchers to capture both the static architecture and dynamic properties of C. aurantiacus EF-Tu, providing insights into its function in protein synthesis and potential adaptations to thermophilic conditions.

How can researchers investigate the interaction between Chloroflexus aurantiacus Elongation factor Tu and the unique components of its translation machinery?

The interaction between EF-Tu and other components of the translation machinery in C. aurantiacus may reveal adaptations specific to this thermophilic photosynthetic organism. A comprehensive investigation requires:

• Binding Partner Identification:

  • Pull-down assays using tagged recombinant EF-Tu

  • Cross-linking coupled with mass spectrometry (XL-MS)

  • Yeast two-hybrid or bacterial two-hybrid screening

  • Comparative interactome analysis with mesophilic EF-Tu homologs

• Quantitative Binding Assays:

  • Surface plasmon resonance (SPR) or bio-layer interferometry (BLI)

  • Microscale thermophoresis (MST) for solution-based measurements

  • Isothermal titration calorimetry (ITC) for thermodynamic parameters

  • Fluorescence anisotropy for labeled ligands

• Functional Translation Assays:

  • Reconstituted in vitro translation system using C. aurantiacus components

  • Single-molecule FRET to monitor EF-Tu dynamics during translation

  • Ribosome binding and GTPase activation assays

  • Temperature-dependent activity measurements

• Structural Studies of Complexes:

  • Cryo-EM of ribosome-EF-Tu-tRNA complexes

  • Crystallization of EF-Tu with binding partners

  • Hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

  • Computational docking validated by mutagenesis

• Temperature-Dependent Interaction Analysis:

  Temperature	Association Rate (kon)	Dissociation Rate (koff)	Affinity (KD)	ΔG	ΔH	TΔS
  25°C	Baseline	Baseline	Baseline	Baseline	Baseline	Baseline
  37°C	↑	↑	Variable	Variable	Variable	Variable
  55°C	↑↑	Variable	Variable	Variable	Variable	Variable
  65°C	Variable	↑↑↑	Variable	Variable	Variable	Variable

This systematic approach would reveal how C. aurantiacus EF-Tu has adapted to function optimally at elevated temperatures and in the context of photosynthetic metabolism, potentially identifying unique features that distinguish it from mesophilic homologs.