Recombinant Desulfovibrio vulgaris Elongation factor G (fusA), partial

What is the structural organization of Desulfovibrio vulgaris EF-G?

Desulfovibrio vulgaris EF-G consists of five domains (I-V), similar to other bacterial elongation factors G. Domain I contains the GTP-binding site and functions as the GTPase domain, while domain IV interacts with the ribosomal A-site during translocation, facilitating the movement of mRNA and tRNA from the A site to the P site on the ribosome . The conformational dynamics between these domains is crucial for proper function . Domain IV is particularly important for the translocation steps as it interacts with the ribosomal A-site, and many mutations affecting EF-G function are found in this region . X-ray crystallography studies have shown that proper interdomain movement is essential for EF-G function, especially for efficient translocation and ribosome recycling .

How does recombinant D. vulgaris EF-G function in protein synthesis?

Recombinant D. vulgaris EF-G, like other bacterial EF-Gs, promotes the translocation step of protein synthesis and participates in ribosome recycling. During translocation, EF-G binds to the ribosome and facilitates the movement of mRNA and tRNA from the A site to the P site, requiring GTP hydrolysis . After protein synthesis termination, EF-G works together with ribosome recycling factor (encoded by frr) to dissociate ribosomes from mRNA . This process is critical for efficient protein synthesis and ribosome availability for new translation cycles. The functional domains of EF-G undergo significant conformational changes during different stages of the translation cycle, which is essential for proper catalytic activity .

What expression systems are recommended for producing recombinant D. vulgaris EF-G?

For producing recombinant D. vulgaris EF-G, E. coli expression systems are commonly used due to their high yield and relative simplicity. When expressing anaerobic bacterial proteins like those from Desulfovibrio, it's important to consider that proper folding may require specific conditions. For optimal expression, BL21(DE3) strains with pET-based vectors under the control of T7 promoter systems are recommended. Induction with 0.1-0.5 mM IPTG at lower temperatures (16-25°C) can improve solubility . Since Desulfovibrio is an anaerobic organism, expression under microaerobic or anaerobic conditions might improve proper folding of the recombinant protein . The addition of specific chaperones may also enhance proper folding, especially when expressing partial constructs of large multidomain proteins like EF-G.

What purification methods are effective for recombinant D. vulgaris EF-G?

Effective purification of recombinant D. vulgaris EF-G typically involves a multi-step process. Initial purification can be achieved using affinity chromatography, especially with nickel-based resins if a His-tag is incorporated into the recombinant construct. For higher purity, additional steps such as ion exchange chromatography (typically on Q-Sepharose) and size exclusion chromatography are recommended . When purifying EF-G, it's important to include GTP or non-hydrolyzable GTP analogs in the buffer to stabilize the protein structure. Typical purification buffers contain 50 mM Tris-HCl (pH 7.5), 100-300 mM KCl, 10 mM MgCl₂, 5-10% glycerol, and 1-5 mM DTT or β-mercaptoethanol to maintain reducing conditions . For anaerobic proteins like those from Desulfovibrio, performing purification steps under reduced oxygen conditions may help maintain proper protein folding and activity.

How can I assess the activity of purified recombinant D. vulgaris EF-G?

The activity of purified recombinant D. vulgaris EF-G can be assessed through several functional assays:

• GTPase activity assay: Measures the hydrolysis of GTP by EF-G, which can be quantified by detecting inorganic phosphate release or through coupled enzyme assays.

• Translocation assay: Assesses the ability of EF-G to promote the movement of tRNA and mRNA on the ribosome, typically using fluorescently labeled tRNAs and FRET-based methods.

• Ribosome recycling assay: Evaluates how efficiently EF-G, along with ribosome recycling factor, can dissociate post-termination ribosome complexes.

• Peptidyl-tRNA drop-off assay: Measures the prevention of premature dissociation of peptidyl-tRNA from the ribosome, which is important for translation fidelity .

In these assays, it's important to include appropriate controls, such as known active EF-G from E. coli and negative controls without EF-G. The kinetic parameters (kcat, KM) obtained from these assays can be compared with literature values to assess the functionality of the recombinant protein .

How do mutations in D. vulgaris fusA affect protein function and antibiotic resistance?

Mutations in the fusA gene can significantly alter EF-G function and contribute to antibiotic resistance. In various bacterial species, mutations in fusA have been associated with resistance to aminoglycosides like kanamycin, gentamicin, and tobramycin . These mutations often occur in domain IV of the protein, which may alter the affinity of aminoglycosides for the ribosomal protein .

Key mutations and their effects include:

Mutations in the switch II region (like F88L) can restrict conformational changes necessary for proper EF-G function, leading to slower translocation and ribosome recycling, as well as increased peptidyl-tRNA drop-off, which results in fitness loss . Secondary mutations (like M16I) can compensate for these fitness defects by partially restoring the conformational dynamics . The mechanism typically involves alterations in the protein's ability to undergo necessary conformational changes during the translocation cycle, affecting its interaction with the ribosome and/or antibiotics.

What strategies can overcome challenges in expressing functional recombinant D. vulgaris EF-G?

Expressing functional recombinant D. vulgaris EF-G presents several challenges due to its large size, multiple domains, and potential toxicity when overexpressed. Advanced strategies to overcome these challenges include:

• Codon optimization: Adjusting the codon usage to match the expression host can significantly improve expression levels. For D. vulgaris proteins expressed in E. coli, this is particularly important as codon preferences differ between these organisms.

• Fusion partners: Using solubility-enhancing fusion partners such as MBP (maltose-binding protein), SUMO, or Thioredoxin can improve folding and solubility of the recombinant protein.

• Expression of individual domains: For structural or functional studies focusing on specific aspects of EF-G, expressing individual domains or combinations of domains can be more successful than expressing the full-length protein.

• Chaperone co-expression: Co-expressing molecular chaperones like GroEL/GroES, DnaK/DnaJ/GrpE, or trigger factor can aid in proper folding of complex multi-domain proteins.

• Cell-free expression systems: For particularly challenging constructs, cell-free expression systems based on E. coli extracts can provide better control over the expression environment and often yield functional protein when in vivo systems fail.

• Anaerobic expression conditions: Since D. vulgaris is an anaerobic organism, expressing its proteins under anaerobic or low-oxygen conditions might yield more properly folded and active protein .

The choice of strategy depends on the specific research question and the particular challenges encountered with the construct of interest.

How can I analyze the conformational dynamics of D. vulgaris EF-G during the translation cycle?

Analyzing the conformational dynamics of D. vulgaris EF-G during the translation cycle requires advanced biophysical techniques:

• Single-molecule FRET (smFRET): By labeling specific residues in different domains of EF-G with fluorescent dyes, the relative movement of these domains during GTP hydrolysis and interaction with the ribosome can be monitored in real-time. This technique has revealed that EF-G undergoes significant conformational changes during translocation.

• Cryo-electron microscopy (cryo-EM): This technique can capture EF-G in different conformational states when bound to the ribosome. Recent advances in cryo-EM have made it possible to achieve near-atomic resolution, providing valuable insights into the structural rearrangements of EF-G during translation.

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): This method can identify regions of EF-G that undergo conformational changes during different stages of the translation cycle by measuring the exchange rate of amide hydrogens with deuterium in the solvent.

• Molecular dynamics simulations: Computational approaches can complement experimental techniques by predicting the conformational changes of EF-G based on crystal structures and providing insights into the energetics of these changes.

• X-ray crystallography of mutant variants: Crystallizing EF-G mutants with altered conformational dynamics (like those affecting antibiotic resistance) can provide structural insights into how these mutations affect protein function .

The conformational dynamics of EF-G are crucial for its function, as demonstrated by studies showing that mutations restricting these dynamics (like F88L) lead to slower translocation and reduced fitness .

What is the role of D. vulgaris EF-G in ribosome recycling and how can this be studied?

D. vulgaris EF-G, like other bacterial EF-Gs, plays a crucial role in ribosome recycling, working in conjunction with ribosome recycling factor (RRF) to dissociate ribosomes from mRNA after the termination of translation . This process is essential for making ribosomes available for new rounds of translation.

To study the role of D. vulgaris EF-G in ribosome recycling:

Understanding the role of EF-G in ribosome recycling is not only important for basic knowledge of translation termination but also has practical applications in biotechnology, as evidenced by the enhanced L-isoleucine production observed in C. glutamicum upon co-overexpression of fusA and frr .

How does D. vulgaris EF-G compare functionally with EF-G from other bacterial species?

D. vulgaris EF-G shares functional similarities with EF-G from other bacterial species but may possess unique characteristics due to the anaerobic lifestyle of Desulfovibrio. Comparative analysis can reveal insights into evolutionary adaptations of the translation machinery.

Functional differences may arise from:

Comparative studies between EF-G from different bacterial species can provide insights into the evolution of the translation machinery and may reveal novel targets for species-specific antibiotic development .

How can recombinant D. vulgaris EF-G be used to study antibiotic resistance mechanisms?

Recombinant D. vulgaris EF-G provides a valuable tool for studying antibiotic resistance mechanisms, particularly those involving translation. Several methodological approaches can be employed:

• Site-directed mutagenesis: By introducing specific mutations identified in resistant clinical isolates (such as those in domain IV or the switch II region) into recombinant D. vulgaris EF-G, researchers can directly assess their impact on antibiotic sensitivity and protein function .

• In vitro translation assays: Reconstituted translation systems using purified components, including mutant EF-G variants, can reveal how specific mutations affect antibiotic inhibition of protein synthesis. These assays can measure the IC50 values of various antibiotics in the presence of wild-type versus mutant EF-G.

• Structural studies of EF-G-antibiotic complexes: X-ray crystallography or cryo-EM of D. vulgaris EF-G in complex with antibiotics can reveal the binding sites and help explain resistance mechanisms . For example, structural analysis has shown how mutations in the switch II region (like F88L) affect fusidic acid binding .

• Fitness cost assessment: Measuring the kinetic parameters of mutant EF-G variants can help explain the fitness costs associated with resistance mutations, such as slower translocation and increased peptidyl-tRNA drop-off observed with the F88L mutation .

• Compensatory mutation analysis: Studying how secondary mutations (like M16I) compensate for the fitness defects of primary resistance mutations (like F88L) can provide insights into the evolution of resistance .

These approaches can help identify new antibiotics targeting EF-G or develop strategies to overcome existing resistance mechanisms, which is particularly important given the role of EF-G mutations in resistance to clinically important antibiotics like aminoglycosides .

What techniques are most effective for analyzing EF-G interactions with the ribosome?

Analyzing EF-G interactions with the ribosome requires sophisticated techniques that can capture transient interactions and conformational changes:

• Cryo-electron microscopy (cryo-EM): Currently the gold standard for visualizing EF-G-ribosome complexes, cryo-EM can achieve near-atomic resolution and capture different states of the translocation process. By stabilizing specific intermediates (using non-hydrolyzable GTP analogs or specific antibiotics), researchers can visualize how EF-G interacts with different parts of the ribosome during the translation cycle.

• Chemical cross-linking coupled with mass spectrometry (XL-MS): This approach identifies interaction points between EF-G and the ribosome by chemically linking residues that are in close proximity, followed by mass spectrometric identification of the cross-linked peptides.

• Förster resonance energy transfer (FRET): By labeling specific sites on EF-G and the ribosome with fluorophore pairs, researchers can monitor changes in their relative positions during translocation. Single-molecule FRET is particularly powerful for detecting intermediate states and measuring the kinetics of conformational changes.

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI): These techniques can measure the binding kinetics and affinity of EF-G for the ribosome under different conditions (GTP vs. GDP, presence of antibiotics, etc.).

• Molecular dynamics simulations: Computational approaches can model the interaction of EF-G with the ribosome based on structural data, providing insights into the energetics and dynamics of the interaction.

• Ribosome footprinting: This technique identifies the ribosomal RNA nucleotides that are protected by EF-G binding, helping to map the interaction surface on the ribosome.

Each of these methods provides complementary information, and combining multiple approaches often yields the most comprehensive understanding of EF-G-ribosome interactions.

How can I troubleshoot issues with recombinant D. vulgaris EF-G activity assays?

When troubleshooting issues with recombinant D. vulgaris EF-G activity assays, several methodological considerations are important:

Issue: Low or No GTPase Activity

Solutions:

• Verify protein folding integrity using circular dichroism or thermal shift assays

• Ensure the presence of adequate Mg²⁺ (typically 5-10 mM) in reaction buffers

• Check for the presence of inhibitory contaminants from purification

• Try different buffer conditions (pH 7.0-8.0, varying salt concentrations)

• Add ribosome to stimulate the GTPase activity of EF-G

Issue: Poor Performance in Translocation Assays

Solutions:

• Ensure all components (ribosomes, tRNAs, mRNA) are active through control experiments

• Optimize the ratio of EF-G to ribosomes (typically 0.5-5 µM EF-G to 0.1-0.2 µM ribosomes)

• Verify that GTP is not limiting or degraded

• Consider using a more sensitive detection method (fluorescence vs. radioactivity)

• Check for factors that might compete with EF-G for ribosome binding

Issue: Inconsistent Results Between Experiments

Solutions:

• Standardize protein concentration determination methods

• Prepare fresh working stocks for each experiment

• Include internal standards and controls in each experiment

• Ensure equipment (spectrophotometers, plate readers) is properly calibrated

• Control for batch-to-batch variation in ribosomes and other components

Issue: Rapid Loss of Activity During Storage

Solutions:

• Store protein at -80°C with glycerol (10-20%)

• Avoid multiple freeze-thaw cycles

• Consider adding stabilizers such as reduced glutathione or DTT

• Store in small aliquots to minimize freeze-thaw damage

• Test different buffer conditions for improved stability

Issue: Discrepancies Between Different Activity Assays

Solutions:

• Ensure that assay conditions are optimized for each specific assay

• Consider that different assays may measure different aspects of EF-G function

• Verify that component concentrations are appropriate for each assay type

• Use appropriate controls to validate each assay system

• Consider that mutations might affect different functions of EF-G to varying degrees

Troubleshooting tables documenting the specific conditions tested and results obtained can be invaluable for systematically addressing issues with activity assays.

What are the implications of D. vulgaris EF-G research for understanding bacterial adaptation to environmental stress?

Research on D. vulgaris EF-G has significant implications for understanding how bacteria adapt their translation machinery to environmental stresses, particularly those relevant to anaerobic environments:

• Adaptation to anaerobic conditions: D. vulgaris, as an anaerobic bacterium, likely has evolved specific features in its translation machinery, including EF-G, to function optimally under anaerobic conditions . Studying these adaptations can reveal how protein synthesis is maintained in oxygen-limited environments.

• Response to nutrient limitation: In environments where D. vulgaris naturally occurs, nutrients can be limiting. EF-G function has been linked to nutrient stress responses through interactions with regulatory molecules like ppGpp . Understanding how D. vulgaris EF-G responds to these signals can provide insights into bacterial survival strategies.

• Metal ion interactions: As a sulfate-reducing bacterium, D. vulgaris occupies niches with unique metal ion compositions. EF-G function depends on metal ions (particularly Mg²⁺), and specific adaptations might exist to maintain function in the presence of sulfides and other compounds found in D. vulgaris' natural environment.

• Translation efficiency under stress: Comparing the kinetic parameters of D. vulgaris EF-G with those from other bacteria under various stress conditions (pH, temperature, salt) can reveal adaptations that maintain protein synthesis under adverse conditions.

• Evolution of antibiotic resistance: Understanding how mutations in D. vulgaris EF-G affect function and confer resistance to antibiotics can provide insights into the evolution of resistance mechanisms in anaerobic bacteria, which might differ from those in aerobic pathogens .

These studies contribute to our understanding of bacterial adaptation at the molecular level and may have applications in biotechnology, particularly for processes involving anaerobic bacteria, such as bioremediation and biofuel production.

How can structural analysis of D. vulgaris EF-G inform the design of novel antibiotics?

Structural analysis of D. vulgaris EF-G can provide valuable insights for the design of novel antibiotics through several approaches:

• Identification of unique binding pockets: Detailed structural analysis can reveal binding pockets that are specific to bacterial EF-G and absent in human translation factors, providing targets for selective antibiotics. Crystal structures of EF-G in different conformational states can identify transient pockets that might be exploited for drug design .

• Structure-based rational drug design: Using the three-dimensional structure of D. vulgaris EF-G, computational approaches can screen virtual libraries of compounds to identify those that might bind to crucial regions of the protein, particularly sites involved in GTP hydrolysis, conformational changes, or ribosome interaction.

• Analysis of resistance mutations: Structural studies of EF-G variants with resistance mutations (like F88L) can reveal how these mutations alter antibiotic binding sites and protein dynamics . This information can guide the design of new compounds that can overcome resistance mechanisms.

• Targeting protein dynamics: Since the conformational dynamics of EF-G are crucial for its function, drugs that interfere with these dynamics could be effective antibiotics. Structural studies combined with molecular dynamics simulations can identify "hot spots" for such intervention .

• Comparative structural analysis: Comparing the structures of EF-G from different bacterial species (including pathogens) can identify conserved features that might serve as broad-spectrum targets as well as species-specific features that could be exploited for narrow-spectrum antibiotics.

• Allosteric inhibitor design: Structural analysis can identify allosteric sites distant from the catalytic center that, when occupied by small molecules, could affect EF-G function through long-range conformational effects. Such allosteric inhibitors might offer advantages over traditional competitive inhibitors.

The integration of structural biology, computational chemistry, and biochemical assays provides a powerful approach for translating structural insights into novel antibiotic candidates targeting bacterial translation.