Recombinant Geobacter sulfurreducens GTPase Der (der)

What is Der GTPase and why is it significant for bacterial research?

Der (also known as EngA) is a unique GTP-binding protein containing two consecutive GTP-binding domains at the N-terminal region. Its homologues are highly conserved in eubacteria but absent in archaea and eukaryotes, making it an excellent antimicrobial target . Der plays an essential role in the biogenesis of 50S ribosomal subunits, with depletion studies showing accumulation of ribosomal subunits and reduction of polysomes and 70S ribosomes . The significance of Der lies in its essentiality for bacterial survival and its potential as a novel antibiotic target, particularly important given the rise of antibiotic resistance . In G. sulfurreducens specifically, studying Der provides insights into ribosome assembly in environmentally important metal-reducing bacteria used in bioremediation applications .

What is the structure and key functional domains of Der GTPase?

Der GTPase from G. sulfurreducens (UniProt: Q74AX4) consists of 438 amino acids with several key domains :

• Two consecutive GTP-binding domains at the N-terminal region, each containing conserved GTP-binding motifs (G1-G5)

• A C-terminal domain involved in ribosome interaction

The protein's sequence contains characteristic GTP-binding motifs including:

• G1/P-loop (GxxxxGKS/T) for nucleotide binding

• G3/Switch II (DxxG) involved in GTP hydrolysis

Temperature-dependent studies in E. coli have shown that both GTP-binding domains are required at low temperature for cell growth, while at high temperature either domain is dispensable, suggesting functional redundancy under certain conditions .

What expression systems work best for recombinant G. sulfurreducens Der protein?

Recombinant G. sulfurreducens Der protein has been successfully expressed in E. coli expression systems . The recommended approach includes:

• Using E. coli BL21(DE3) or similar strains with T7 expression systems

• Incorporating an N-terminal or C-terminal His-tag for purification purposes

• Optimizing expression temperature (typically 16-18°C) to enhance solubility

• Using rich media such as Terrific Broth supplemented with glucose

The entire coding sequence (residues 1-438) should be included to ensure proper protein folding and function . Commercial preparations typically achieve purity >85% as verified by SDS-PAGE .

How can I verify the functional integrity of purified Der protein?

Functional verification of purified Der GTPase should include:

• GTPase activity assay measuring phosphate release from GTP hydrolysis

• Ribosome binding assay using sucrose density gradient centrifugation to confirm specific interaction with 50S ribosomal subunits in the presence of GTP analogues (particularly GMPPNP)

• Size exclusion chromatography to confirm proper oligomeric state

• Circular dichroism to verify correct secondary structure folding

Crucially, Der interacts with 50S ribosomal subunits specifically in the presence of GTP analogues, and this GTP-dependent ribosome association is a key functional characteristic to verify .

How does Der GTPase contribute to ribosome assembly in bacteria?

Der plays a critical role in 50S ribosomal subunit biogenesis. Studies in E. coli have demonstrated:

• Der associates specifically with 50S ribosomal subunits in a GTP-dependent manner

• Depletion of Der results in accumulation of structurally unstable 50S subunits that dissociate into aberrant subunits at lower Mg²⁺ concentrations

• Der-depleted cells accumulate precursors of both 23S and 16S rRNAs

• Der depletion leads to reduced polysomes and 70S ribosomes with concurrent accumulation of 50S and 30S ribosomal subunits

These findings suggest Der functions as a ribosomal assembly factor, potentially acting as a quality control checkpoint to ensure proper 50S subunit maturation before it engages in translation. The GTP-dependent binding indicates Der may assess ribosomal conformation through GTP hydrolysis cycles.

What approaches can be used to study the dual GTPase domains of Der?

Studying the dual GTPase domains requires sophisticated experimental approaches:

• Site-directed mutagenesis to selectively inactivate each domain:

  • G1 motif (K→A) mutations to abolish nucleotide binding

  • G3 motif (D→N) mutations to prevent GTP hydrolysis while maintaining binding

• Domain-specific activity analysis:

  • Comparing GTPase activity of wild-type protein versus single-domain mutants

  • Assessing temperature-dependent requirements of each domain

  • Measuring nucleotide binding affinities for each domain separately

• Structural studies:

  • Crystallography or cryo-EM with different nucleotides bound

  • FRET experiments to monitor interdomain conformational changes

Research has shown an intriguing temperature dependence in E. coli, where both domains are essential at low temperatures while either domain is sufficient at higher temperatures . This suggests complex functional interplay between the domains that requires careful experimental design to elucidate.

How can Der GTPase be targeted for antibiotic development?

Der GTPase represents a promising antibiotic target due to several favorable characteristics:

• It is essential and ubiquitous in bacteria but absent in eukaryotes

• It has a well-defined enzymatic activity that can be screened against

• Crystal structures (such as from T. maritima Der) provide templates for structure-based drug design

Successful approaches have included:

• Virtual screening of chemical libraries against Der GTPase structure, which identified 257 potential inhibitors in one study

• In vitro assessment of compounds for Der GTPase inhibition

• In vivo testing for antibacterial activity

Three structurally diverse compounds (SBI-34462, SBI-34566, and SBI-34612) have been identified as both enzymatic inhibitors of Der GTPase and biologically active against bacterial cells . These compounds provide scaffolds for further development of novel antibiotics targeting antibiotic-resistant bacteria.

The GTP-binding site is a primary target for inhibitor design, with successful compounds typically interacting with residues involved in GTP binding and hydrolysis .

What genetic approaches can be used to study Der function in G. sulfurreducens?

The development of genetic systems for G. sulfurreducens enables sophisticated in vivo studies of Der:

• Antibiotic sensitivity profiling has been established for G. sulfurreducens, enabling selection of appropriate markers

• Electroporation protocols for introducing foreign DNA into G. sulfurreducens have been optimized

• Two classes of broad-host-range vectors (IncQ and pBBR1) can replicate in G. sulfurreducens

• The IncQ plasmid pCD342 has been established as a suitable expression vector for G. sulfurreducens

For studying essential genes like Der, conditional approaches are necessary:

• Regulatable promoters to control Der expression levels

• Temperature-sensitive mutants to study domain requirements

• Depletion strains that gradually reduce Der protein levels

All genetic manipulations must account for G. sulfurreducens being an anaerobic organism, requiring appropriate handling under oxygen-free conditions .

What are the optimal conditions for measuring Der GTPase activity in vitro?

Optimal conditions for measuring Der GTPase activity include:

• Buffer components:

  • HEPES or Tris buffer (pH 7.5-8.0)

  • Magnesium chloride (5-10 mM), essential for GTPase function

  • Potassium chloride (50-100 mM)

  • Reducing agent (DTT or β-mercaptoethanol, 1-5 mM)

• Reaction parameters:

  • Temperature: 30-37°C (note that temperature affects domain requirements)

  • GTP concentration: Typically 0.1-1 mM

  • Protein concentration: 0.1-1 μM

• Detection methods:

  • Malachite green assay for measuring released phosphate

  • HPLC-based methods for nucleotide analysis

  • Coupled enzymatic assays using phosphate detection

Activity measurements should assess both the kinetics (kcat, Km) and the effects of ribosomal components, as Der's interaction with 50S ribosomal subunits may alter its catalytic properties .

How should researchers design experiments to study Der-ribosome interactions?

Studying Der-ribosome interactions requires careful experimental design:

• Sucrose density gradient centrifugation:

  • Der specifically associates with 50S subunits only in the presence of GMPPNP (a non-hydrolyzable GTP analogue)

  • Magnesium concentration is critical, as Der-depleted 50S subunits are unstable at lower Mg²⁺ concentrations

• Pull-down assays:

  • Using His-tagged Der to pull down associated ribosomal components

  • Analysis of co-precipitated rRNA and ribosomal proteins

• Cryo-EM analysis:

  • Visualization of Der binding site on the 50S subunit

  • Structural changes in ribosome conformation upon Der binding

• In vivo approaches:

  • Ribosome profiling before and after Der depletion

  • Analysis of rRNA processing patterns in Der-depleted cells

All experiments should control for nucleotide state (GDP, GTP, or analogues) and magnesium concentration, as these factors significantly influence Der-ribosome interactions .

What are the key considerations for long-term storage of recombinant Der protein?

Proper storage of recombinant G. sulfurreducens Der protein is critical for maintaining activity:

• Storage conditions:

  • Store at -20°C/-80°C to maintain stability

  • Liquid form has a typical shelf life of 6 months at -20°C/-80°C

  • Lyophilized form maintains stability for approximately 12 months at -20°C/-80°C

• Buffer components for stability:

  • Addition of 5-50% glycerol (final concentration) helps prevent freeze-thaw damage

  • Inclusion of reducing agents protects cysteine residues

  • GTP or non-hydrolyzable analogues may stabilize protein conformation

• Handling recommendations:

  • Avoid repeated freeze-thaw cycles

  • Store working aliquots at 4°C for up to one week

  • Centrifuge vials briefly before opening to bring contents to the bottom

• Reconstitution guidelines:

  • Reconstitute lyophilized protein in deionized sterile water to 0.1-1.0 mg/mL

  • Add glycerol to a final concentration of 50% for long-term storage

How can researchers address challenges in comparing Der function across different bacterial species?

Comparative analysis of Der across bacterial species presents several challenges:

• Experimental standardization:

  • Use consistent buffer conditions and assay methods

  • Account for optimal temperature differences between species

  • Normalize activity data to account for expression level differences

• Complementation studies:

  • Express G. sulfurreducens Der in Der-depleted E. coli or other bacteria

  • Assess functional conservation and species-specific requirements

  • Compare ribosome binding properties across species

• Sequence and structure analysis:

  • Compare conserved motifs versus species-specific variations

  • Analyze correlation between sequence divergence and functional differences

  • Identify species-specific insertions/deletions that may confer specialized functions

• Environmental context:

  • Consider G. sulfurreducens' anaerobic lifestyle and metal-reducing capabilities

  • Analyze Der expression and modification under different growth conditions

The unique ecological niche of G. sulfurreducens as a metal-reducing bacterium may have led to specific adaptations in Der function compared to model organisms like E. coli.

What approaches help resolve discrepancies between in vitro and in vivo Der functional data?

Discrepancies between in vitro and in vivo results are common and require systematic troubleshooting:

• Potential causes of discrepancies:

  • In vitro conditions may not recapitulate the cellular environment

  • Der may require specific ribosomal components for full activity

  • Post-translational modifications present in vivo may be absent in recombinant protein

  • Temperature-dependent effects may differ between controlled in vitro and variable cellular environments

• Resolution strategies:

  • Test activity in more complex in vitro systems that better mimic cellular conditions

  • Include ribosomal components in in vitro assays

  • Compare wild-type versus domain mutants both in vitro and in vivo

  • Analyze Der's relationship with stress response pathways

• Genetic approaches:

  • Create point mutations based on in vitro findings and test in vivo

  • Use temperature-sensitive mutants to correlate in vitro temperature effects with in vivo phenotypes

  • Develop depletion strains with tunable Der expression levels

Understanding the relationship between Der function and stress response pathways, such as those involving RelGsu in G. sulfurreducens , may help explain context-dependent activity differences.

How might Der function integrate with stress response pathways in G. sulfurreducens?

The relationship between Der and stress response pathways represents an important research frontier:

• G. sulfurreducens contains a RelA homolog (RelGsu) that regulates ppGpp synthesis and degradation in response to nutrient limitation

• ppGpp is a key regulatory molecule in the stringent response affecting ribosome synthesis

• Der function in ribosome assembly may intersect with the ppGpp-mediated stress response

Research approaches to explore this connection could include:

• Analyzing Der activity and ribosome profiles during RelGsu-mediated stress responses

• Studying Der expression and modification under nutrient limitation

• Investigating potential direct interactions between Der and components of stress response pathways

• Examining how metal-reducing conditions affect Der function and stress responses

The intersection of ribosome assembly, GTPase function, and stress response represents a promising area for understanding bacterial adaptation to environmental challenges.

What techniques will advance structure-function studies of Der GTPase?

Several emerging technologies hold promise for deeper understanding of Der structure-function relationships:

• Cryo-electron microscopy for:

  • Visualizing Der-ribosome complexes at near-atomic resolution

  • Capturing different conformational states during the GTPase cycle

  • Identifying the precise binding interface on the 50S subunit

• Single-molecule approaches for:

  • Measuring GTPase activity of individual Der molecules

  • Observing Der-ribosome interaction dynamics in real-time

  • Detecting conformational changes between the two GTPase domains

• In-cell studies for:

  • Tracking Der localization and dynamics in living bacteria

  • Measuring ribosome assembly kinetics with and without Der

  • Observing temperature-dependent changes in Der function

These advanced techniques will help resolve the molecular mechanism of how Der's dual GTPase domains coordinate ribosome assembly and quality control in bacteria.