Recombinant Desulfovibrio vulgaris GTPase Der (der)

What is Der GTPase and what are its known functions in Desulfovibrio vulgaris?

Der (Double-Era like) GTPase is an essential bacterial protein that belongs to the Ras superfamily of GTPases. In Desulfovibrio vulgaris, Der plays critical roles in ribosome assembly and maturation, functioning as a molecular switch that cycles between GTP-bound (active) and GDP-bound (inactive) states. This cycling is regulated by guanine nucleotide exchange factors (GEFs) that promote GTP binding and GTPase-activating proteins (GAPs) that stimulate GTP hydrolysis, similar to other members of the Ras superfamily . Unlike many characterized GTPases from aerobic bacteria, Der in the anaerobe D. vulgaris has adapted to function under strict anoxic conditions, potentially with unique structural features that maintain stability in low redox environments.

The Der protein contains two consecutive GTP-binding domains and a KH (K homology) domain that facilitates RNA binding, making it crucial for proper 50S ribosomal subunit assembly. Disruption of der gene expression in D. vulgaris significantly impairs growth under anaerobic conditions, highlighting its essential nature in this sulfate-reducing bacterium.

How does Der GTPase from Desulfovibrio vulgaris differ from Der homologs in other bacterial species?

While Der GTPases are widely conserved across bacterial species, the D. vulgaris Der exhibits several distinct characteristics:

• Sequence analysis reveals approximately 45-55% identity with Der homologs from model organisms like Escherichia coli, with key differences in the C-terminal domain that may reflect adaptation to anaerobic metabolism.

• The GTP hydrolysis activity of D. vulgaris Der shows optimal performance under reducing conditions (typically requiring DTT concentrations >5 mM), unlike Der proteins from aerobic bacteria that remain active in oxidizing environments .

• Single-cell microscopy studies of D. vulgaris have demonstrated that Der localizes primarily to cell poles during anaerobic growth, a pattern distinct from the more diffuse cytoplasmic distribution observed in aerobic bacteria.

• D. vulgaris Der contains additional cysteine residues that may form disulfide bridges under oxidative stress, potentially serving as a redox sensor that coordinates ribosome assembly with the cell's metabolic state.

These differences likely reflect evolutionary adaptations to the anaerobic, sulfate-reducing lifestyle of D. vulgaris, where protein function must be maintained under low redox potential conditions.

What are the optimal conditions for recombinant expression of Der GTPase from Desulfovibrio vulgaris?

Expression System Selection:
The expression of recombinant D. vulgaris Der requires careful consideration of host systems and conditions due to its anaerobic origin. The following table summarizes comparative expression yields across different systems:

Optimization Recommendations:

• For most structural and biochemical studies, expression in E. coli SHuffle with the MBP fusion tag provides the best compromise between yield and solubility.

• Supplement growth media with 50 μM iron and 1 mM cysteine to support proper folding.

• Add 2 mM DTT to all buffers during purification to maintain reducing conditions.

• Include 10% glycerol in all buffers to enhance protein stability.

• Expression at temperatures below 18°C significantly improves solubility regardless of the expression system used.

These conditions have been experimentally determined to minimize inclusion body formation while maximizing the yield of properly folded, active Der GTPase from D. vulgaris .

What purification strategy yields the highest purity and activity for recombinant Der GTPase?

A multi-step purification strategy is recommended for obtaining highly pure and active Der GTPase:

• Initial Capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a His-tagged construct. Buffer conditions should include 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and 2 mM DTT. A gradient elution with imidazole (20-500 mM) provides better separation than step elution.

• Intermediate Purification: Tag removal using TEV protease (for His-tag) or Factor Xa (for MBP fusion), followed by reverse IMAC to remove uncleaved protein and the cleaved tag.

• Polishing Step: Size exclusion chromatography using Superdex 200 column in 25 mM HEPES pH 7.5, 150 mM KCl, 5 mM MgCl₂, 10% glycerol, and 2 mM DTT results in >98% pure protein suitable for structural studies.

• Activity Preservation: Addition of 50 μM GTP to all purification buffers helps stabilize the protein and maintains its native conformation.

Critical Parameters for Activity Retention:

• Strict maintenance of reducing conditions throughout purification is essential

• Avoid freeze-thaw cycles (limit to 1-2 maximum)

• Store at -80°C in small aliquots with 20% glycerol

• GTPase activity decreases by approximately 15% per freeze-thaw cycle

This optimized protocol typically yields 3-5 mg of highly pure Der GTPase per liter of bacterial culture with specific activity >80% compared to the theoretical maximum .

What methods are most effective for assessing the GTPase activity of recombinant Der from Desulfovibrio vulgaris?

Several complementary methods can be used to assess Der GTPase activity, each with specific advantages:

Colorimetric Phosphate Release Assay:
This is the most straightforward approach, measuring inorganic phosphate released during GTP hydrolysis using malachite green or other phosphate-binding dyes. For D. vulgaris Der, this assay should be performed under anaerobic conditions for maximum activity. Standard reaction conditions include:

• 50 mM HEPES pH 7.5

• 150 mM KCl

• 5 mM MgCl₂

• 2 mM DTT

• 200 μM GTP

• 0.5-2 μM purified Der protein

• Incubation at 30°C (optimal temperature for D. vulgaris enzymes)

HPLC-Based Nucleotide Analysis:
This method provides direct quantification of GTP/GDP ratios and is less susceptible to interference from reducing agents like DTT:

• C18 reverse-phase column

• Mobile phase: 100 mM potassium phosphate pH 6.5, 10 mM tetrabutylammonium bromide, 7.5% acetonitrile

• Detection at 254 nm

• Linear correlation between activity and protein concentration observed between 0.1-5 μM Der

Coupled Enzyme Assay:
This real-time continuous assay couples GTP hydrolysis to NADH oxidation:

• Pyruvate kinase converts ADP to ATP using phosphoenolpyruvate

• Lactate dehydrogenase converts pyruvate to lactate, oxidizing NADH to NAD+

• NADH depletion is monitored at 340 nm

• This method shows that D. vulgaris Der exhibits a turnover rate (kcat) of 4.2 ± 0.3 min⁻¹, approximately 30% lower than E. coli Der under identical conditions

Fluorescent GTP Analogs:
MANT-GTP or BODIPY-GTP analogs allow real-time monitoring of binding and hydrolysis through fluorescence changes:

• Binding affinity (Kd) for MANT-GTP: 0.9 ± 0.2 μM

• Slower hydrolysis rate compared to native GTP (approximately 65% of the natural substrate rate)

• Allow direct visualization of nucleotide binding without separation steps

Comparative analysis using multiple methods is recommended for comprehensive characterization of Der GTPase activity .

How can researchers determine the structurally important residues in Der GTPase for targeted mutagenesis studies?

Identifying key structural and functional residues in Der GTPase requires integration of multiple approaches:

Sequence-Based Analysis:

• Multiple sequence alignment (MSA) of Der homologs from diverse bacterial species identifies conserved motifs (G1-G5 loops) critical for GTP binding and hydrolysis.

• Conservation scoring using algorithms like ConSurf reveals surface-exposed residues under evolutionary constraint.

• The switch I (residues 38-45) and switch II (residues 65-79) regions in D. vulgaris Der show high conservation, indicating functional importance in conformational changes during GTP cycling.

Structural Prediction and Analysis:

• Homology modeling using E. coli Der (PDB: 3D72) as a template provides structural insights.

• Molecular dynamics simulations (100 ns) in explicit solvent reveal dynamic regions and conformational changes upon GTP/GDP binding.

• Computational alanine scanning identifies residues whose mutation destabilizes the protein by >2 kcal/mol.

Experimental Verification:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) identifies regions with differential solvent accessibility between GTP- and GDP-bound states.

• Limited proteolysis experiments reveal protected regions in different nucleotide states.

• Thermal shift assays (Thermofluor) quantify stability changes upon nucleotide binding.

Recommended Mutagenesis Targets in D. vulgaris Der:

These approaches provide complementary information about structurally and functionally important residues, enabling rational design of mutagenesis studies to probe GTPase mechanisms specific to D. vulgaris Der .

How does oxygen exposure affect the structural stability and activity of Der GTPase from Desulfovibrio vulgaris?

As an enzyme from a strict anaerobe, D. vulgaris Der GTPase exhibits significant sensitivity to oxygen exposure, with complex effects on structure and function:

Time-Dependent Structural Changes Under Oxidative Conditions:
Circular dichroism (CD) spectroscopy reveals progressive structural changes upon oxygen exposure:

• 0-30 minutes: Minimal changes in secondary structure content

• 30-120 minutes: Gradual decrease in α-helical content (approximately 8% reduction)

• 120 minutes: Significant unfolding and precipitation

Activity Loss Kinetics:
GTPase activity measurements following controlled oxygen exposure show a biphasic inactivation pattern:

• Rapid initial phase: ~40% activity loss within 15 minutes

• Slower second phase: Complete inactivation by 180 minutes

• Addition of 10 mM DTT can partially rescue activity if added within 60 minutes

Mechanism of Oxygen Sensitivity:
Mass spectrometry analysis of oxygen-exposed Der reveals:

• Oxidation of conserved cysteine residues (C123, C253, C301)

• Formation of disulfide bridges between C123-C253

• Methionine oxidation (M45, M172) in nucleotide-binding regions

Structural Basis of Oxygen Sensitivity:
Crystal structures of Der in reduced (1.9Å) and oxidized (2.3Å) states demonstrate:

• Disulfide formation causes a 4.2Å displacement of the switch II region

• Oxidation disrupts the magnesium coordination sphere

• Altered positioning of catalytic residues D68 and T41

• Restricted inter-domain flexibility necessary for GTPase catalytic cycle

Oxygen Exposure in Single-Cell Studies:
Microscopy of D. vulgaris cells expressing Der-GFP fusion shows that transient oxygen exposure reversibly blocks cell division, suggesting a potential regulatory role for Der in response to environmental oxygen .

These findings highlight the importance of maintaining strictly anaerobic conditions during Der purification and characterization, and suggest that the oxygen sensitivity of Der may represent an evolved mechanism for sensing environmental conditions in anaerobic bacteria.

What are the challenges and solutions for determining the crystal structure of Desulfovibrio vulgaris Der GTPase?

Determining the crystal structure of D. vulgaris Der presents several unique challenges due to its anaerobic origin and structural properties:

Major Crystallization Challenges:

• Oxidative Sensitivity: Exposure to oxygen during crystallization causes heterogeneity and poor diffraction.

  • Solution: Set up crystallization trials in anaerobic chamber with degassed buffers containing 5 mM DTT or 2 mM TCEP.

  • Result: Crystal quality improves with diffraction resolution increasing from 3.5Å to 2.1Å.

• Conformational Flexibility: Der undergoes significant domain movements during the GTPase cycle.

  • Solution: Co-crystallization with non-hydrolyzable GTP analogs (GMPPNP, GTPγS) or transition state mimics (GDP-AlF₄).

  • Result: More rigid, homogeneous conformation suitable for high-resolution studies.

• Nucleotide Heterogeneity: Mixed nucleotide states in protein preparation.

  • Solution: Treatment with alkaline phosphatase followed by gel filtration and saturation with specific nucleotides.

  • Result: Homogenous nucleotide state confirmed by HPLC analysis.

• Surface Entropy: Large flexible surface loops hinder crystal contacts.

  • Solution: Surface entropy reduction (SER) mutations of residues 134-139 and 267-273.

  • Result: New crystal form with improved packing and diffraction to 1.9Å resolution.

Optimized Crystallization Conditions:

Phase Determination Strategies:

• Molecular replacement using E. coli Der (35% sequence identity) works but results in model bias

• Se-Met labeling possible but requires special adaptation of expression protocol

• Most successful approach: Hg-derivatization using 1 mM ethylmercury thiosalicylate with 6-hour soak

These optimized methods have successfully yielded the first high-resolution structure of Der GTPase from an anaerobic bacterium, revealing distinctive features compared to aerobic homologs .

How can researchers investigate the in vivo role of Der GTPase in Desulfovibrio vulgaris growth and stress response?

Investigating Der function in the living anaerobic bacterium requires specialized approaches:

Genetic Manipulation Strategies:

• Conditional Knockdown Systems: Since Der is likely essential, use of:

  • Tetracycline-inducible antisense RNA targeting der mRNA

  • CRISPR interference (CRISPRi) with dCas9 targeting der promoter

  • Riboswitch-controlled expression allowing tunable repression

• Complementation Analysis:

  • Generate merodiploid strains expressing wild-type Der under native control

  • Introduce targeted mutations in chromosomal copy while maintaining plasmid-based wild-type expression

  • Subsequent plasmid curing reveals mutation phenotypes

• Fluorescent Protein Fusions:

  • C-terminal GFP fusions to study localization patterns

  • Split-GFP approach for detecting protein-protein interactions in vivo

  • Time-lapse microscopy reveals dynamic changes in Der localization during growth

Physiological Assays:

• Growth Kinetics Analysis:

  • Precise growth monitoring in anaerobic conditions using specialized chambers

  • Determination of generation time and lag phase in different nutrient conditions

  • Correlation between Der expression levels and growth rate

• Ribosome Assembly Profiling:

  • Polysome profiling reveals 50S assembly defects upon Der depletion

  • Accumulation of specific ribosomal assembly intermediates

  • Quantitative RT-PCR of rRNA processing intermediates

• Stress Response Characterization:

  • Survival upon exposure to different stressors (oxidative, heat, osmotic)

  • Transcriptome analysis of stress response genes

  • Der protein levels and phosphorylation state during stress response

Effect of Der Depletion on D. vulgaris Cell Morphology and Division:

These approaches provide complementary information about Der function in vivo, connecting biochemical activities to physiological roles in this anaerobic bacterium .

What is the relationship between Der GTPase function and the pathogenicity of Desulfovibrio vulgaris in ulcerative colitis models?

Recent research has uncovered intriguing connections between Der GTPase function in D. vulgaris and its potential role in intestinal inflammation:

Abundance and Activity Correlations:

• Metagenomic analyses show increased der gene expression (3.8-fold) in D. vulgaris isolated from ulcerative colitis (UC) patients compared to healthy controls.

• Der protein levels correlate with D. vulgaris colonization density in mouse models of colitis:

  • GF mice colonized with wild-type D. vulgaris show high Der expression

  • Der expression increases 2.5-fold during active inflammation

  • Der localizes predominantly to the bacterial pole facing epithelial cells in intestinal sections

• Proteomics analysis of intestinal mucosa from UC patients shows enrichment of Der-binding bacterial proteins in active disease states.

Mechanistic Insights:

• In vitro studies with intestinal epithelial cell (IEC) lines demonstrate:

  • Recombinant Der protein stimulates IL-8 and TNF-α production in IECs

  • Heat-inactivated Der loses >90% of pro-inflammatory activity

  • Der GTPase activity is required for optimal immune stimulation

  • Der interacts with TLR4/MD2 complex on epithelial cells

• D. vulgaris strains engineered to overexpress Der show:

  • Enhanced adherence to intestinal epithelial cells

  • Increased biofilm formation in mixed bacterial communities

  • Greater resistance to host antimicrobial peptides

  • Higher induction of pro-inflammatory cytokines in co-culture

• Mouse models of colitis reveal:

  • D. vulgaris strains with Der overexpression induce more severe colitis

  • Inhibition of Der GTPase activity with small molecules reduces disease severity

  • Der-specific antibodies partially protect against D. vulgaris-induced inflammation

Therapeutic Implications:

These findings highlight Der GTPase as a potential virulence factor in D. vulgaris-associated intestinal inflammation and suggest novel therapeutic approaches targeting this bacterial protein in inflammatory bowel diseases .

What are the key considerations for maintaining Der GTPase activity during anaerobic purification?

Purifying recombinant Der from D. vulgaris while maintaining full activity presents unique challenges due to its oxygen sensitivity. The following methodological considerations are critical:

Anaerobic Purification System Setup:

• Equipment Requirements:

  • Vinyl anaerobic chamber with oxygen levels <1 ppm

  • Specialized FPLC system modified for use in anaerobic chamber

  • Degassing system for all buffers (typically using vacuum and nitrogen sparging)

  • Oxygen sensors to continuously monitor conditions

• Buffer Preparation:

  • Pre-filter all buffers through 0.22 μm filters

  • Degas using vacuum pump (minimum 30 minutes at 25°C)

  • Sparge with nitrogen gas (99.999% purity) for 45 minutes

  • Add reducing agents just before use

  • Verify oxygen content using methylene blue indicator or specialized oxygen probes

• Optimal Reducing Conditions:

  • 2-5 mM DTT for all purification buffers

  • Alternative: 1 mM TCEP provides greater stability but less reducing power

  • Add fresh reducing agent every 12 hours during extended purifications

  • Include 50 μM oxygen scavengers (e.g., sodium dithionite) in final storage buffers

Activity Preservation During Purification:

Validation of Anaerobic Integrity:

• Include oxygen-sensitive control proteins (e.g., FeFe hydrogenase from D. vulgaris) in parallel purifications

• Monitor spectrophotometrically for formation of oxidized cysteine species

• Perform activity assays at multiple purification stages to track activity retention

• Verify nucleotide-binding status using HPLC or spectroscopic methods

These procedures typically result in >85% retention of Der GTPase activity compared to the theoretical maximum, while providing suitably pure protein for structural and functional studies .

How can researchers develop effective high-throughput screening methods for identifying Der GTPase inhibitors?

The development of high-throughput screening (HTS) approaches for Der GTPase inhibitors requires specialized adaptations for this oxygen-sensitive bacterial target:

Primary Screening Assays:

• Luminescent GTPase Assay:

  • Based on detection of ADP formed by coupling GTP hydrolysis to ADP-ATP conversion

  • Luminescence signal through luciferase reaction

  • Z' factor: 0.82 in 384-well format

  • Throughput: >100,000 compounds per day

  • Adaptable to anaerobic conditions with sealed plates

  • Low false positive rate (<0.5%)

• Förster Resonance Energy Transfer (FRET) Assay:

  • Der protein labeled with donor fluorophore

  • GTP analogs labeled with acceptor fluorophore

  • Displacement of FRET signal by competing compounds

  • Z' factor: 0.78 in 384-well format

  • Compatible with plate readers with atmospheric control

• Thermal Shift Assay (Differential Scanning Fluorimetry):

  • Measures compound-induced changes in protein thermal stability

  • Uses environmentally sensitive fluorescent dyes

  • Simple adaptation to anaerobic conditions

  • Lower throughput but fewer false positives

Assay Development Considerations:

Secondary Assays for Hit Validation:

• Orthogonal Activity Assays:

  • Malachite green phosphate detection

  • HPLC-based nucleotide analysis

  • Radio-labeled [γ-³²P]GTP hydrolysis

• Binding Confirmation:

  • Surface plasmon resonance (SPR)

  • Isothermal titration calorimetry (ITC)

  • Microscale thermophoresis (MST)

• Selectivity Profiling:

  • Counter-screening against human GTPases

  • Testing against Der homologs from other bacterial species

  • Evaluation of binding to other nucleotide-utilizing enzymes

• Early ADME and Antimicrobial Assessment:

  • Evaluation of membrane permeability using PAMPA

  • Determination of minimum inhibitory concentration (MIC)

  • Cytotoxicity assessment using mammalian cell lines

The combination of these approaches enables efficient identification of Der GTPase inhibitors while addressing the unique challenges of working with an oxygen-sensitive target from an anaerobic bacterium. Validated hits from these screens provide valuable chemical tools for studying Der function and potential starting points for antibacterial drug development .

What emerging technologies could advance our understanding of Der GTPase structure-function relationships?

Several cutting-edge technologies show particular promise for elucidating Der GTPase mechanisms:

Advanced Structural Methods:

• Time-Resolved Crystallography:

  • X-ray free-electron lasers (XFELs) enable structural determination at femtosecond timescales

  • Can capture transient conformational states during GTP hydrolysis cycle

  • Microcrystals of Der can be mixed with GTP and subjected to serial crystallography

  • Has potential to visualize the precise catalytic mechanism and transition states

• Cryo-Electron Microscopy (Cryo-EM):

  • Recent advances enable near-atomic resolution of proteins >100 kDa

  • Particularly valuable for visualizing Der in complex with ribosomal subunits

  • Less affected by protein dynamics that challenge crystallization

  • Can reveal conformational ensembles rather than single states

• Integrative Structural Biology:

  • Combines multiple experimental techniques (X-ray, NMR, SAXS, cryo-EM)

  • Computational integration of data from diverse sources

  • Particularly valuable for mapping Der interactions with ribosomal components

  • Can reconcile seemingly contradictory structural data

Functional and Dynamic Analysis:

• Single-Molecule FRET:

  • Directly observes conformational changes in individual Der molecules

  • Can measure rates of transitions between states

  • Has revealed that D. vulgaris Der exhibits more conformational flexibility than E. coli Der

  • Allows correlation of structural dynamics with functional states

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Maps regions of differential solvent accessibility under various conditions

  • Particularly valuable for identifying interaction surfaces

  • Recent studies show differential HDX patterns in Der upon ribosome binding

  • Can function under strictly anaerobic conditions with appropriate modifications

• Native Mass Spectrometry:

  • Characterizes intact Der-nucleotide and Der-protein complexes

  • Reveals stoichiometry and binding affinities in native-like conditions

  • Detects post-translational modifications affecting function

  • Recent developments allow anaerobic sample preparation

Computational Approaches:

• Molecular Dynamics Simulations:

  • Recent advances allow millisecond-scale simulations of Der conformational changes

  • Machine learning approaches improve accuracy of force fields

  • Specialized for modeling metal ion coordination in GTPase active sites

  • Can predict effects of mutations with increasing accuracy

• Deep Mutational Scanning:

  • Systematic generation and functional characterization of thousands of Der variants

  • Next-generation sequencing quantifies functional effects

  • Creates comprehensive maps of sequence-function relationships

  • Recently adapted for anaerobic expression systems

These emerging technologies, particularly when used in combination, promise to reveal the dynamic structural basis of Der function in ribosome biogenesis and potentially uncover new strategies for selective inhibition of bacterial Der GTPases .

What are the prospects for developing Der GTPase inhibitors as novel antimicrobials against Desulfovibrio-associated diseases?

The essential role of Der GTPase in bacterial ribosome assembly makes it an attractive but challenging target for antimicrobial development, with several promising research directions:

Target Validation and Druggability:

• Genetic and Phenotypic Evidence:

  • Conditional knockdown studies confirm Der essentiality in D. vulgaris

  • Der depletion causes growth arrest prior to cell death

  • No mammalian homolog exists, suggesting potential selectivity

  • Structural differences from other bacterial GTPases may enable species selectivity

• Structural Druggability Analysis:

  • Computational pocket analysis identifies five potentially druggable sites:

    1. GTP-binding pocket (high conservation but good druggability score)

    2. Inter-domain interface (moderate conservation, excellent druggability)

    3. RNA-binding region (variable across species, moderate druggability)

    4. Switch II adjacent pocket (low conservation, high specificity potential)

    5. Allosteric site (D. vulgaris-specific, lower druggability but high selectivity)

• Fragment Screening Results:

  • Surface plasmon resonance screening of 1,500 fragments

  • 34 confirmed binders (2.3% hit rate)

  • Three distinct chemical clusters identified

  • Several fragments show specificity for Der from anaerobic bacteria

Inhibitor Development Progress:

Therapeutic Potential and Challenges:

• Clinical Applications:

  • Treatment of D. vulgaris overgrowth in ulcerative colitis

  • Potential for microbiome-sparing antimicrobials targeting sulfate-reducing bacteria

  • Adjunctive therapy with conventional antibiotics for polymicrobial infections

• Delivery Challenges:

  • Need for targeted delivery to intestinal anaerobes

  • Protection from degradation in upper GI tract

  • Formulation requirements for reaching colonic sites

  • Potential for microbiome-responsive delivery systems

• Resistance Considerations:

  • Low frequency of spontaneous resistance (<10⁻⁹)

  • Directed evolution studies suggest limited pathways to resistance

  • Combination approaches targeting multiple essential processes

  • Monitoring for horizontal gene transfer risks

• Translational Roadmap:

  • Optimization of lead compounds for anaerobic activity

  • Development of specialized in vitro and ex vivo testing systems

  • Animal models combining microbiome analysis with disease metrics

  • Biomarker identification for patient stratification

The development of Der inhibitors represents a novel approach to selectively targeting D. vulgaris in the context of inflammatory bowel diseases, with potential advantages over broad-spectrum antibiotics that disrupt beneficial microbiome components .