Recombinant Desulfovibrio vulgaris Elongation factor Ts (tsf)

How does the structure of Desulfovibrio vulgaris EF-Ts differ from EF-Ts in other bacterial species?

The Desulfovibrio vulgaris EF-Ts shares structural features with other bacterial EF-Ts proteins but has distinctive sequence characteristics. While the core function of nucleotide exchange is conserved across species, the specific amino acid sequence can vary significantly. Phylogenetic analyses of similar proteins in other bacterial species reveal evolutionary divergence that affects interactions with partner proteins .

For effective comparative studies, researchers should consider:

• Performing multiple sequence alignments with EF-Ts from model organisms like E. coli

• Analyzing conserved domains across species using structural prediction tools

• Examining differences in key regions that interact with EF-Tu

• Evaluating how these structural differences might affect functional properties

Research approaches might include circular dichroism (CD) spectroscopy to analyze secondary structural elements and X-ray crystallography or cryo-EM to determine high-resolution structures that can be compared with known structures from other bacterial species.

What are the optimal conditions for expression and purification of recombinant D. vulgaris EF-Ts?

For optimal expression and purification of recombinant D. vulgaris EF-Ts, researchers should consider the following methodological approach based on established protocols:

• Expression System: Transformation of the recombinant plasmid into E. coli BL21(DE3)pLysS strain has shown good results for EF-Ts expression . Alternative expression hosts may include E. coli BL21 cells transformed with appropriate vector plasmids.

• Growth Conditions:

  • Culture in Luria-Bertani media at 37°C until OD600 reaches approximately 0.7

  • Induce protein expression with 1 mM IPTG and ZnSO4

  • Continue expression at 15°C for 24 hours to maximize yield while minimizing inclusion body formation

• Purification Strategy:

  • Use N-terminal His6-tag fusion for affinity chromatography

  • Employ a sequential purification approach including immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography

  • Consider including an ion exchange chromatography step if higher purity is required

• Storage Conditions:

  • For lyophilized form: store at -20°C/-80°C (shelf life up to 12 months)

  • For liquid form: store at -20°C/-80°C (shelf life approximately 6 months)

  • For working aliquots: store at 4°C for up to one week

  • Avoid repeated freeze-thaw cycles

How can researchers assess the functional activity of purified recombinant D. vulgaris EF-Ts?

To assess the functional activity of purified recombinant D. vulgaris EF-Ts, researchers can employ several complementary assays that evaluate its nucleotide exchange activity and interaction with EF-Tu:

• Nucleotide Exchange Assay:

  • Pre-load EF-Tu with fluorescent GDP analogs (such as mant-GDP)

  • Monitor the rate of nucleotide exchange by measuring the decrease in fluorescence signal when the labeled GDP is displaced by unlabeled GTP

  • Measure this exchange rate in the presence and absence of purified EF-Ts to quantify its catalytic efficiency

• Ternary Complex Formation Assay:

  • Use fluorescently labeled aa-tRNA (such as Cy3-acp3U47-labeled Phe-tRNAPhe)

  • Track fluorescence intensity changes as a function of EF-Tu concentration in the presence and absence of EF-Ts

  • Calculate apparent affinity constants under different conditions

• Steady-State Measurements:

  • Titrate either EF-Tu·GTP or EF-Tu·EF-Ts complex into a reaction mixture containing fluorescently labeled aa-tRNA and GTP

  • Observe EF-Tu-dependent increase in fluorescence intensity (typically plateauing at ~30% above baseline)

  • Determine and compare dissociation constants (KD values) as shown in the following table :

How does EF-Ts interact with EF-Tu to regulate protein synthesis in bacterial systems?

EF-Ts interacts with EF-Tu through a complex mechanism that regulates protein synthesis in bacterial systems:

• Nucleotide Exchange Function:

  • EF-Ts catalyzes the exchange of GDP for GTP on EF-Tu, which is essential for ternary complex formation

  • This exchange is necessary because EF-Tu has approximately 60-fold higher affinity for GDP over GTP and exhibits slow spontaneous nucleotide exchange

  • The process involves a conformational change in EF-Tu that is accelerated by EF-Ts

• Direct Facilitation of Ternary Complex Dynamics:

  • EF-Ts not only facilitates nucleotide exchange but also directly affects the formation and disassociation of the EF-Tu·GTP·aa-tRNA ternary complex

  • Studies have shown that both formation and decay rates of ternary complex are accelerated in the presence of EF-Ts

  • EF-Ts attenuates the affinity of EF-Tu for GTP and destabilizes ternary complex in the presence of non-hydrolyzable GTP analogs

• Conformational Control:

  • The interaction involves a nucleotide-dependent, rate-determining conformational change in EF-Tu

  • This conformational change is significantly accelerated by EF-Ts, which serves as a regulatory mechanism for controlling ternary complex abundance

• Complex Formation:

  • Gel filtration studies show that EF-Tu and EF-Ts form a stable one-to-one complex

  • This interaction is critical for the rapid cycling of EF-Tu between its GDP- and GTP-bound states

What role does EF-Ts play in bacterial stress response and proteostasis networks?

Recent research has revealed that EF-Ts plays significant roles in bacterial stress response and proteostasis networks beyond its canonical function in translation:

• Interaction with Chaperone Networks:

  • EF-Ts has been found to influence the stability of EF-Tu, which in turn affects how cells respond to stress conditions

  • In heat shock conditions, the stability of EF-Tu is modulated by a sophisticated network of molecular chaperones, including EF-Ts, to regulate protein biosynthesis

• Regulation under Stress Conditions:

  • During stress, the EF-Tu:EF-Ts interaction becomes part of a larger protein quality control (PQC) process

  • This system helps ensure proteostasis in cells under stressed conditions by attenuating protein biosynthesis, which can be harmful to cell survival in adverse environments

• Interactions with Other Chaperones:

  • EF-Ts shows functional interactions with other chaperones like Hsp33 and trigger factor (TF)

  • While EF-Tu unfolding and aggregation can be induced by Hsp33, this process is still evident even when EF-Tu is in a complex state with EF-Ts

  • Interestingly, though TF alone has little effect on EF-Tu stability, it can markedly amplify Hsp33-mediated EF-Tu unfolding and aggregation

• Synergistic Chaperone Activity:

  • The interactions between EF-Ts, EF-Tu, Hsp33, and TF constitute an example of synergistic unfoldase/aggregase activity of molecular chaperones

  • This suggests that EF-Ts participates in a complex regulatory network that modulates translation in response to cellular stress

How can researchers use recombinant D. vulgaris EF-Ts in structural and functional studies of protein translation systems?

Researchers can employ recombinant D. vulgaris EF-Ts in various sophisticated structural and functional studies to advance understanding of protein translation systems:

• Cryo-EM and X-ray Crystallography Studies:

  • Use purified recombinant EF-Ts in complex with EF-Tu to determine high-resolution structures

  • Analyze conformational changes during different stages of nucleotide exchange

  • Compare structural features with EF-Ts from other bacterial species to identify conserved and divergent regions

• Single-Molecule FRET Experiments:

  • Label EF-Ts and EF-Tu with fluorescent dyes at specific positions

  • Monitor real-time conformational changes during their interaction

  • Measure kinetics of association and dissociation under various conditions

  • Study the effect of nucleotides (GTP, GDP, non-hydrolyzable analogs) on complex formation and stability

• Reconstituted Translation Systems:

  • Incorporate recombinant D. vulgaris EF-Ts into in vitro translation systems

  • Evaluate its ability to support protein synthesis compared to EF-Ts from model organisms

  • Analyze the kinetics of translation and effects on accuracy under different conditions

  • Investigate species-specific aspects of translation factor function

• Crosslinking and Mass Spectrometry:

  • Use chemical crosslinking followed by mass spectrometry analysis to map interaction interfaces

  • Identify key residues involved in protein-protein interactions

  • Compare interaction patterns with those of EF-Ts from other bacterial species

What approaches can be used to investigate the evolutionary divergence of EF-Ts across bacterial species?

To investigate the evolutionary divergence of EF-Ts across bacterial species, researchers can employ several complementary approaches:

• Comparative Genomics and Phylogenetic Analysis:

  • Construct phylogenetic trees of EF-Ts sequences from diverse bacterial species

  • Identify patterns of conservation and divergence in key functional domains

  • Analyze co-evolution with interacting partners like EF-Tu

  • Map sequence changes to functional differences across bacterial lineages

• Structure-Function Relationship Studies:

  • Perform alanine scanning mutagenesis of EF-Ts to identify critical residues

  • Create chimeric proteins by swapping domains between EF-Ts from different species

  • Analyze how specific amino acid substitutions affect function and interaction networks

  • Use techniques like trajectory-scanning mutagenesis to trace evolutionary pathways

• Specificity Determination:

  • Study how specificity residues in EF-Ts have changed across bacterial lineages

  • Examine the impact of these changes on interactions with EF-Tu

  • Investigate whether evolutionary changes have led to altered specificity profiles

  • Determine if paralogous expansions in some bacterial lineages have resulted in functional divergence

• Adaptive Evolution Analysis:

  • Look for signatures of positive selection in EF-Ts sequences

  • Identify sites under selective pressure that may contribute to functional adaptation

  • Investigate whether mutations that prevent crosstalk with other cellular components have enabled the expansion and diversification of this protein family

What are common challenges in working with recombinant D. vulgaris EF-Ts and how can they be addressed?

Researchers working with recombinant D. vulgaris EF-Ts often encounter several challenges that can be addressed through specific troubleshooting approaches:

• Protein Solubility Issues:

  • Challenge: D. vulgaris proteins may exhibit poor solubility when expressed in E. coli systems

  • Solution: Optimize expression conditions by lowering induction temperature (15°C is recommended), using solubility-enhancing fusion tags, or adding solubility enhancers to the culture medium

  • Alternative approach: Use specialized E. coli strains designed for expression of challenging proteins

• Protein Stability Concerns:

  • Challenge: Recombinant EF-Ts may show reduced stability during storage or experimental procedures

  • Solution: Add stabilizing agents such as glycerol (5-50%, with 50% being the default recommendation) to storage buffers

  • Best practice: Aliquot the purified protein to avoid repeated freeze-thaw cycles and store at -80°C for long-term storage or at 4°C for up to one week for working aliquots

• Activity Assessment Complications:

  • Challenge: Difficulties in accurately measuring nucleotide exchange activity

  • Solution: Implement multiple complementary assays, including direct measurement of nucleotide exchange rates and functional assessment of ternary complex formation

  • Control experiment: Include both positive controls (e.g., E. coli EF-Ts) and negative controls in activity assays

• Protein-Protein Interaction Characterization:

  • Challenge: Detecting and quantifying interactions between EF-Ts and partner proteins like EF-Tu

  • Solution: Use a combination of techniques including gel filtration chromatography, isothermal titration calorimetry, and fluorescence-based assays

  • Advanced approach: Implement FRET-based assays using fluorescently labeled proteins to monitor interactions in real-time

How can researchers address inconsistencies in functional assay results when studying D. vulgaris EF-Ts?

When confronted with inconsistencies in functional assay results for D. vulgaris EF-Ts, researchers should implement a systematic troubleshooting approach:

• Protein Quality Assessment:

  • Verify protein purity using multiple methods (SDS-PAGE, mass spectrometry)

  • Confirm protein integrity by checking for degradation products

  • Assess protein aggregation state using analytical size exclusion chromatography or dynamic light scattering

  • Use circular dichroism to verify proper folding of the recombinant protein

• Assay Conditions Optimization:

  • Systematically test different buffer compositions, pH values, and ionic strengths

  • Evaluate the effect of different divalent cations (Mg²⁺, Mn²⁺, Ca²⁺) on activity

  • Optimize protein and substrate concentrations to ensure linear response ranges

  • Control temperature precisely during assays to minimize variability

• Methodological Considerations:

  • Implement different detection methods to cross-validate results (e.g., fluorescence-based assays versus radioactive assays)

  • Consider time-resolved measurements to capture transient states or conformational changes

  • Use stopped-flow techniques for fast kinetic measurements if applicable

  • Establish proper controls for background signal and non-specific interactions

• Data Analysis Refinements:

  • Apply appropriate kinetic models that account for complex formation mechanisms

  • Use global fitting approaches when analyzing multiple datasets

  • Account for potential cooperativity or allosteric effects in protein-protein interactions

  • Consider using Bayesian statistical approaches for more robust parameter estimation when data is noisy

How might D. vulgaris EF-Ts be involved in bacterial stress response mechanisms?

Recent research suggests that D. vulgaris EF-Ts plays significant roles in bacterial stress response mechanisms beyond its canonical function in translation:

• Integration with Chaperone Networks:

  • D. vulgaris EF-Ts likely participates in proteostasis networks similar to those observed in other bacterial species

  • It may interact with stress-response proteins like Hsp33, which has been shown to exhibit unfoldase and aggregase activity against EF-Tu in its reduced state

  • The interaction between EF-Ts, EF-Tu, and other chaperones could form a regulatory network that responds to various stress conditions

• Regulation of Protein Synthesis Under Stress:

  • By modulating EF-Tu activity, EF-Ts may help attenuate protein biosynthesis during stress, which can be beneficial for cell survival

  • This regulation could be part of a larger protein quality control process that ensures proteostasis in cells under stressed conditions

  • The apparently contradictory function of some chaperones (promoting client misfolding) might actually be linked to these regulatory processes

• Response to Oxidative and Redox Stress:

  • Given the interaction with redox-regulated chaperones like Hsp33, EF-Ts may play a role in cellular responses to oxidative stress

  • The stability of the EF-Tu:EF-Ts complex might be influenced by redox conditions in the cell

  • This would allow for rapid modulation of translation efficiency in response to changing environmental conditions

• Potential Role in Biofilm Formation and Survival:

  • D. vulgaris is known for its ability to form biofilms and survive in challenging environments

  • Translation factors including EF-Ts may contribute to stress adaptation mechanisms that enable biofilm persistence

  • Research examining the expression and activity of EF-Ts under biofilm versus planktonic conditions could reveal novel functions

What potential applications exist for using recombinant D. vulgaris EF-Ts in synthetic biology and biotechnology?

Recombinant D. vulgaris EF-Ts offers several promising applications in synthetic biology and biotechnology:

• Enhanced Cell-Free Protein Synthesis Systems:

  • Incorporation of D. vulgaris EF-Ts into cell-free protein synthesis systems may enhance translation efficiency

  • This could be particularly valuable for the production of difficult-to-express proteins

  • The unique properties of D. vulgaris EF-Ts might offer advantages in specific reaction conditions, such as anaerobic or high-stress environments

• Biosensor Development:

  • The interaction between EF-Ts and EF-Tu could be harnessed to develop biosensors for specific environmental conditions

  • By linking their interaction to reporter systems, researchers could monitor cellular stress responses

  • This approach could be useful for detecting environmental toxins that affect translation machinery

• Biofuel Production Enhancement:

  • D. vulgaris is relevant to biofuel research due to its anaerobic metabolism and sulfate-reducing capabilities

  • Engineering strains with modified EF-Ts might improve protein expression under stress conditions relevant to biofuel production

  • This could contribute to more efficient biocatalyst development for converting waste materials to biofuels

• Antibiotic Research Applications:

  • As translation factors are targets for several classes of antibiotics, D. vulgaris EF-Ts could be used in screening platforms for novel antimicrobials

  • The unique features of D. vulgaris EF-Ts compared to other bacterial species might provide insights for species-specific antibiotic development

  • Understanding how antibiotics like thiostrepton, GE2270A, and kirromycin affect the EF-Tu:EF-Ts interaction could lead to new therapeutic approaches