Recombinant GTPase obg (obg)

What is the structural organization of Obg proteins?

Obg proteins possess a unique tripartite structure with distinct functional domains. The highly conserved N-terminal domain (NTD) is glycine-rich and demonstrates significant potential for protein-protein interactions. The central region contains the conserved GTP-binding domain with characteristic G1-G5 motifs essential for nucleotide binding and hydrolysis. The C-terminal domain is more variable across species and contributes to specific functional adaptations .

Structurally, the N-terminal domain of bacterial ObgE serves as a tRNA structural mimic, as revealed by cryo-electron microscopy studies of the 50S·ObgE·GMPPNP complex. This domain makes specific interactions with the peptidyl-transferase center, displaying remarkable similarity to Class I release factors. These structural characteristics enable Obg proteins to interact with ribosomes and potentially regulate translation during stress responses .

The G-domains of Obg contain the classic G1 (Walker A motif [GxxxxGK(S/T)]), G2 (switch I [xTx]), G3 (switch II or Walker B motif [DxxG]), G4 [(N/T)(K/Q)xD], and G5 [SA(K/L)] motifs, which collectively coordinate nucleotide binding and hydrolysis. These conserved sequence elements are crucial for the protein's GTPase activity and subsequent biological functions .

How do Obg proteins differ from other GTPases in their biochemical properties?

Unlike classic Ras-like GTPases, Obg proteins exhibit distinct biochemical characteristics that set them apart as specialized cellular sensors. Obg GTPases display slow rates of GTP hydrolysis with micromolar binding constants for both GTP and GDP. They also demonstrate rapid dissociation constants for nucleotides that are 10³-10⁵ times faster than those observed with Ras-like GTPases .

Conventional GTPases typically exhibit high affinities for nucleotides, low dissociation rates without exchange factors, and low intrinsic hydrolysis activity. Their activities are regulated by guanine exchange factors (GEFs), GTPase activating proteins (GAPs), and guanidine dissociation inhibitors (GDIs). In contrast, Obg proteins function as intracellular sensors with their nucleotide-bound state primarily controlled by the relative GTP/GDP concentration rather than accessory regulatory proteins .

The functional association between Obg and ribosomes is also distinctive. ObgE has been demonstrated to be a ribosome-dependent GTPase. Upon binding to guanosine tetraphosphate (ppGpp)—the global regulator of stringent response—ObgE exhibits enhanced interaction with the 50S ribosomal subunit, resulting in increased equilibrium dissociation of the 70S ribosome into subunits .

What are the known functions of Obg proteins in bacterial cells?

Obg proteins are multifunctional GTPases implicated in several essential cellular processes. In bacteria, they serve as critical regulators of ribosome assembly, participating in the maturation of the 50S ribosomal subunit. ObgE acts as an anti-association factor that prevents premature ribosomal subunit association and downstream steps in translation by binding to the 50S subunit .

Beyond ribosome biogenesis, Obg proteins play important roles in stress responses. They function as molecular switches during the stringent response, with their activity modulated by the alarmone ppGpp. This interaction suggests Obg may act as a checkpoint in the final stages of 50S subunit assembly under normal growth conditions while also regulating 50S subunit production and translation participation under stressed conditions .

Obg homologs have been found to be essential for the survival of both Gram-positive and Gram-negative bacteria, including Bacillus subtilis, Streptococcus coelicolor, Staphylococcus pneumoniae, S. aureus, Haemophilus influenzae, Caulobacter crescentus, Escherichia coli, Vibrio harveyi, and V. cholerae. This universal essentiality across bacterial species underscores their fundamental importance in bacterial physiology .

What experimental approaches are most effective for studying Obg GTPase activity in vitro?

Several complementary approaches have been optimized for investigating Obg GTPase activity in vitro. The BIOMOL Green assay has been established as an effective primary screening method for measuring Obg GTPase activity by detecting released phosphate resulting from GTP hydrolysis. This colorimetric assay is based on the principle that malachite green complexed with phosphomolybdate leads to a spectral shift that can be quantified .

For optimal assay conditions with recombinant Obg proteins, incubation with GTP (250 μM) in the presence of Mg²⁺ for 18 hours at 37°C provides the most favorable signal window (approximately 4.25). The dependence on Mg²⁺ for GTP binding and hydrolysis is a critical factor, with maximal formation of Obg-GTP complexes occurring between 5 and 10 mM Mg²⁺. In contrast, GDP binding by Obg doesn't require Mg²⁺ and is actually inhibited at concentrations above 1 mM .

Secondary activity assessment methods utilize fluorescent N-methyl-3'-O-anthranoyl-(mant)-guanine nucleotide analogs, such as mant-GTP and mant-GDP. These fluorescent analogs enable real-time monitoring of nucleotide binding and exchange, providing complementary information to the endpoint phosphate detection assays. Kinetic characterization of Obg proteins typically reveals Michaelis-Menten constants (Km) in the micromolar range (e.g., 78 μM for Obg from N. gonorrhoeae) .

How can researchers generate and characterize Obg mutants for functional studies?

Generation of Obg mutants for functional studies involves strategic targeting of key domains and motifs. For G-domain mutations, researchers should focus on the conserved G1-G5 motifs that coordinate nucleotide binding and hydrolysis. Multiple alterations in these domains can be introduced using site-directed mutagenesis techniques to create variants unable to bind nucleotides, which serve as valuable controls in biochemical assays .

A methodical approach for creating recombinant Obg constructs begins with PCR amplification of the target gene using primers containing appropriate restriction sites. For example, to create C-terminal 6×His fusion proteins, primers can be designed with NcoI and HindIII restriction sites to facilitate cloning into expression vectors like pET-28a. DNA sequencing should be performed to confirm the presence of desired mutations and the absence of unintended alterations .

For expression and purification of recombinant Obg proteins, E. coli BL21(DE3) strains are commonly used. Cultures should be grown to mid-logarithmic phase (OD600 of 0.5) before induction with IPTG (typically 0.5 mM) for 3-4 hours at 30°C. For optimal solubility, purification should be performed using nickel affinity chromatography with buffers containing 50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM MgCl2, and varying concentrations of imidazole for washing (20-40 mM) and elution (250-300 mM) .

What is the current understanding of Obg's role in ribosome assembly?

Obg plays a critical role in ribosome assembly, particularly in the maturation of the large ribosomal subunit. Detailed studies have revealed that ObgE in E. coli functions as an anti-association factor that prevents ribosomal subunit association by binding to the 50S subunit. The cryo-EM structure of the 50S·ObgE·GMPPNP complex indicates that the evolutionarily conserved N-terminal domain of ObgE mimics tRNA structure and makes specific interactions with the peptidyl-transferase center .

Current models suggest that Obg may act as a checkpoint in the final stages of 50S subunit assembly under normal growth conditions, ensuring that only properly matured subunits participate in translation. Under stress conditions, Obg's binding to ppGpp enhances its interaction with the 50S subunit, increasing the equilibrium dissociation of 70S ribosomes into subunits. This mechanism might represent an underrecognized means of translation control by environmental cues .

In mitochondria, the Obg homolog GTPBP5 is essential for the formation of functional mitoribosomal large subunits (mtLSU). Loss of human GTPBP5 severely affects the formation of 55S mitoribosomes, resulting in immature mtLSU particles lacking the bL36m protein and containing an excess of assembly factors like GTPBP7, GTPBP10, MALSU1, and MTERF4. This ultimately attenuates mitochondrial translation and oxidative phosphorylation function .

How can researchers effectively target Obg for antimicrobial drug development?

Targeting Obg for antimicrobial development requires a systematic approach beginning with high-throughput screening (HTS) assays. The BIOMOL Green assay can be optimized for HTS by establishing proper positive and negative controls. Negative controls should consist of complete reaction mixtures, while positive controls should include reactions lacking Mg²⁺, which is essential for GTP hydrolysis. This setup provides a reliable signal window for detecting potential inhibitors .

The selection of GTP concentration is critical for inhibitor screening. While using substrate concentrations at or below the Km of the enzyme (typically 70-80 μM for Obg) creates conditions sensitive to competitive inhibitors, higher GTP concentrations may provide a wider signal window to identify diverse inhibitors. This approach acknowledges Obg's proposed intracellular action as a GTP sensor whose activity depends on GTP/GDP binding affinity under different Mg²⁺ concentrations .

Follow-up assays should include dose-dependence studies for promising compounds and secondary screening methods based on the binding of fluorescent nucleotide analogs like mant-GTP and mant-GDP. To assess the broad-spectrum potential of inhibitors, comparative testing should be performed against Obg proteins from multiple clinically relevant pathogens. For instance, recombinant versions of Obg proteins from Klebsiella pneumoniae and methicillin-resistant Staphylococcus aureus can be used to confirm inhibitory activity across species .

What is the relationship between Obg and stress response mechanisms?

Obg proteins serve as critical mediators between stress response mechanisms and cellular physiology. A key interaction occurs between ObgE and ppGpp, the global regulator of stringent response. Upon binding to ppGpp, ObgE exhibits enhanced interaction with the 50S ribosomal subunit, resulting in increased equilibrium dissociation of 70S ribosomes. This mechanism potentially allows bacteria to rapidly adjust translation in response to nutrient limitation and other stresses .

The dual role of Obg in both ribosome assembly and stress response suggests it functions as an integration point for environmental signals and cellular physiology. Under normal growth conditions, Obg participates in ribosome assembly checkpoints, while under stress conditions, its altered activity may redirect cellular resources by modulating translation efficiency. This function positions Obg as a regulatory factor that helps cells adapt to changing environmental conditions .

In bacteria, Obg GTPases might couple ribosome assembly with growth control pathways by sensing the cellular GTP/GDP ratio. This sensing capability allows cells to coordinate ribosome production—a major energy investment—with nutrient availability and growth rate. The slow GTPase activity and rapid nucleotide exchange properties of Obg make it particularly suited for this sensor role, as its nucleotide-bound state will reflect intracellular GTP/GDP levels rather than being locked in one conformation .

What are the best protocols for purifying active recombinant Obg proteins?

Purification of active recombinant Obg proteins requires careful attention to buffer conditions and protein solubility. The following optimized protocol yields highly pure and active protein:

• Construct preparation: Clone the obg gene into an expression vector with a C-terminal 6×His tag. For example, amplify the gene using primers containing appropriate restriction sites (NcoI and HindIII) and clone into pET-28a vector .

• Expression conditions: Transform the construct into E. coli BL21(DE3) and grow cultures in LB medium with appropriate antibiotics at 37°C until OD600 reaches 0.5. Induce protein expression with 0.5 mM IPTG and continue incubation at 30°C for 3-4 hours to minimize inclusion body formation .

• Cell lysis: Harvest cells by centrifugation and resuspend in lysis buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, 10 mM MgCl2, 10% glycerol, 1 mM PMSF, and 10 mM imidazole. Lyse cells by sonication or using a French press followed by centrifugation at 16,000×g for 30 minutes to remove cell debris .

• Affinity purification: Apply the clarified lysate to Ni-NTA agarose equilibrated with binding buffer. Wash extensively with binding buffer containing 20-40 mM imidazole to remove non-specifically bound proteins. Elute the target protein with elution buffer containing 250-300 mM imidazole .

• Buffer exchange and storage: Dialyze the eluted protein against storage buffer (50 mM HEPES pH 7.5, 200 mM KCl, 10 mM MgCl2, 5 mM DTT, 10% glycerol) to remove imidazole. Concentrate as needed using ultrafiltration devices with appropriate molecular weight cutoffs. Store aliquots at -80°C to maintain activity .

Using this protocol, researchers can expect yields of 5-10 mg of active Obg protein per liter of bacterial culture with >90% purity as assessed by SDS-PAGE.

How can researchers accurately measure Obg-ribosome interactions?

Measuring Obg-ribosome interactions requires specialized techniques that can detect these dynamic associations. A comprehensive approach combines multiple complementary methods:

• Pre-steady state fast kinetics: This approach allows researchers to monitor the rapid kinetics of Obg-ribosome binding and its effects on subunit association. Using stopped-flow fluorescence spectroscopy with fluorescently labeled ribosomal subunits, investigators can measure the anti-association activity of Obg in real-time. This technique revealed that ObgE prevents ribosomal subunit association by binding to the 50S subunit .

• Equilibrium binding assays: Techniques like sucrose density gradient centrifugation can be used to assess the equilibrium binding of Obg to ribosomal subunits and its impact on 70S ribosome stability. This approach demonstrated that ObgE binding to ppGpp enhances its interaction with the 50S subunit, resulting in increased dissociation of 70S ribosomes into subunits .

• Cryo-electron microscopy (cryo-EM): High-resolution structural analysis by cryo-EM provides detailed insights into Obg-ribosome complexes. This method was instrumental in revealing that the N-terminal domain of ObgE is a tRNA structural mimic that makes specific interactions with the peptidyl-transferase center of the 50S subunit .

• Filter binding assays: Quantitative assessment of Obg binding to ribosomes can be performed using radiolabeled Obg or nucleotides and filter binding approaches. This technique allows measurement of binding affinities and kinetics under various conditions, including the presence of different nucleotides or stress molecules like ppGpp .

• Ribosome profiling: To study Obg-ribosome interactions in vivo, ribosome profiling can be employed to monitor ribosome occupancy on mRNAs in wild-type versus Obg-depleted or mutant cells. This approach provides insights into how Obg affects translation in living cells .

What analytical methods are most suitable for studying Obg nucleotide binding properties?

Several analytical methods have been optimized for characterizing the unique nucleotide binding properties of Obg proteins:

• Fluorescent nucleotide analogs: Using N-methyl-3'-O-anthranoyl-(mant)-guanine nucleotide analogs (mant-GTP and mant-GDP) allows real-time monitoring of nucleotide binding and exchange. These fluorescent probes exhibit increased fluorescence intensity upon binding to Obg proteins, enabling kinetic measurements of association and dissociation rates .

• Isothermal titration calorimetry (ITC): ITC provides direct measurement of binding thermodynamics, including binding constants (Kd), enthalpy changes (ΔH), entropy changes (ΔS), and binding stoichiometry. This technique revealed that Obg GTPases have micromolar binding constants for both GTP and GDP, distinguishing them from Ras-like GTPases with nanomolar affinities .

• Surface plasmon resonance (SPR): SPR allows label-free, real-time monitoring of Obg-nucleotide interactions with high sensitivity. By immobilizing Obg proteins on sensor chips and flowing nucleotides at various concentrations, researchers can determine association and dissociation rate constants as well as equilibrium binding constants .

• Filter binding assays: Quantitative assessment of nucleotide binding can be performed using radiolabeled nucleotides (³²P-GTP or ³²P-GDP) and nitrocellulose filter binding. This approach is particularly useful for measuring the effects of different conditions (pH, salt, Mg²⁺ concentration) on nucleotide binding .

• UV cross-linking: This technique uses UV irradiation to covalently link bound nucleotides to Obg proteins, allowing assessment of nucleotide binding capacity under various conditions. Combined with SDS-PAGE and autoradiography or phosphorimaging, this method provides a straightforward approach for qualitative binding studies .

How should researchers interpret conflicting data regarding Obg function?

Interpreting conflicting data regarding Obg function requires systematic analysis of experimental conditions and context-dependent factors:

• Organism-specific differences: Compare results across different bacterial species, as Obg functions may have diverged despite structural conservation. For instance, while Obg proteins universally participate in ribosome assembly, their specific interactions with stress response pathways might vary between organisms .

• Growth conditions: Analyze whether conflicting results stem from different growth or stress conditions. Obg function is highly context-dependent, acting differently under normal growth versus stringent response conditions. Results obtained under different nutrient availability, temperature, or stress conditions may legitimately reflect different aspects of Obg activity .

• Experimental approaches: Evaluate methodological differences that might explain conflicting results. In vitro biochemical assays may not fully recapitulate the complex environment of living cells, while genetic approaches might reveal phenotypes resulting from multiple perturbed pathways rather than direct Obg function .

• Protein concentration effects: Consider whether different concentrations of Obg in various experiments might explain discrepancies. As a nucleotide sensor, Obg activity depends on both its concentration and the GTP/GDP ratio, meaning that overexpression or depletion experiments might yield apparently contradictory results .

• Multifunctionality: Recognize that seemingly conflicting data may actually reflect Obg's multifunctional nature. As both a ribosome assembly factor and stress response mediator, Obg simultaneously participates in multiple cellular processes. Different experimental setups might emphasize one function over another without contradiction .

What are common pitfalls in GTPase activity assays and how can they be avoided?

GTPase activity assays for Obg proteins present several technical challenges that require careful consideration:

• Magnesium dependency: A critical factor in Obg GTPase activity assays is Mg²⁺ concentration. Obg proteins show maximal formation of GTP complexes between 5-10 mM Mg²⁺, while GDP binding is inhibited above 1 mM Mg²⁺. Assays should include appropriate Mg²⁺ controls, as absence of Mg²⁺ effectively abolishes GTP hydrolysis activity .

• Incubation conditions: The slow GTPase activity of Obg proteins necessitates extended incubation periods. Optimization experiments revealed that 18-hour incubation at 37°C provides the optimal signal window for detecting activity differences. Shorter incubations may fail to detect subtle changes in activity, while longer periods might increase background signal .

• Substrate concentration: GTP concentration significantly impacts assay sensitivity to different inhibitor types. While using substrate concentrations at or below the Km (typically 70-80 μM for Obg) creates conditions sensitive to competitive inhibitors, higher GTP concentrations provide a wider signal window. Researchers should select concentrations based on their specific objectives .

• Buffer composition: GTPase activity is sensitive to buffer composition, particularly pH and salt concentration. Optimized conditions typically include 50 mM HEPES pH 7.5 and 200 mM KCl, but systematic optimization for each specific Obg protein is recommended. Additionally, reducing agents like DTT (1-5 mM) help maintain protein stability during extended incubations .

• Spontaneous GTP hydrolysis: Background GTP hydrolysis in the absence of enzyme must be carefully controlled. Include enzyme-free controls and subtract their values from experimental readings. Additionally, fresh GTP stocks should be prepared regularly to minimize the presence of pre-existing free phosphate .

How can researchers resolve difficulties in expressing soluble Obg proteins?

Expression of soluble, active Obg proteins presents challenges that can be addressed through systematic optimization:

• Expression temperature: Lowering the expression temperature to 30°C or even 25°C often dramatically improves Obg solubility by slowing protein synthesis and allowing more time for proper folding. This modification alone can increase soluble protein yield by 2-3 fold compared to standard 37°C expression .

• Induction conditions: Reducing IPTG concentration from the standard 1 mM to 0.1-0.5 mM decreases expression rate and improves folding. Additionally, inducing at higher cell densities (OD600 of 0.6-0.8 rather than 0.3-0.5) often yields more soluble protein .

• Fusion tags: N-terminal fusion partners like maltose-binding protein (MBP), NusA, or SUMO can significantly enhance Obg solubility. These larger tags promote proper folding and can be removed post-purification using specific proteases. The standard C-terminal 6×His tag is sufficient for purification but offers minimal solubility enhancement .

• Lysis buffer optimization: Including stabilizing additives in lysis buffers improves Obg recovery. Key additives include glycerol (10-15%), which stabilizes hydrophobic interactions; non-ionic detergents like 0.1% Triton X-100, which reduce protein aggregation; and nucleotides (50-100 μM GTP or GDP), which stabilize the native conformation .

• Coexpression with chaperones: Coexpression with bacterial chaperone systems like GroEL/GroES or DnaK/DnaJ/GrpE can dramatically improve Obg folding and solubility. Commercially available chaperone plasmids compatible with pET expression systems enable easy implementation of this strategy .

What are emerging techniques that could advance Obg research?

Several cutting-edge technologies show particular promise for advancing our understanding of Obg proteins:

• Cryo-electron tomography: This technique could extend our understanding of Obg-ribosome interactions by visualizing these complexes within their native cellular environment. While cryo-EM has provided valuable insights into Obg-ribosome complexes in vitro, cryo-electron tomography would reveal how these interactions occur in the context of the crowded cellular milieu and potentially identify additional interaction partners .

• Single-molecule techniques: Methods like single-molecule FRET (smFRET) and optical tweezers could illuminate the dynamics of Obg-ribosome interactions and nucleotide exchange at unprecedented resolution. These approaches would reveal transient states and conformational changes that are obscured in ensemble measurements, providing mechanistic insights into how Obg functions as a molecular switch .

• Time-resolved structural studies: Techniques like time-resolved X-ray crystallography and time-resolved cryo-EM could capture structural snapshots of Obg throughout its GTPase cycle, revealing how nucleotide binding and hydrolysis trigger conformational changes that mediate its biological functions .

• Ribosome profiling and selective ribosome profiling: These techniques could provide genome-wide insights into how Obg affects translation of specific mRNAs under various conditions. By identifying which genes are most sensitive to Obg activity, researchers could better understand its role in coordinating the stress response with translational regulation .

• Proximity labeling approaches: Methods like BioID or APEX2 could identify the dynamic interactome of Obg under different cellular conditions. By tagging Obg with these enzymatic labels, researchers could capture both stable and transient interaction partners, potentially revealing new functional connections between Obg and other cellular pathways .

What unexplored aspects of Obg function warrant further investigation?

Despite significant advances in understanding Obg proteins, several important aspects remain underexplored:

• Post-translational modifications: The potential regulation of Obg through post-translational modifications like phosphorylation, acetylation, or methylation remains largely unexplored. These modifications could fine-tune Obg activity in response to specific cellular signals beyond nucleotide binding .

• Interaction with small RNAs: While Obg's interaction with ribosomes is well-established, its potential binding to small regulatory RNAs has not been thoroughly investigated. Given the tRNA-mimicking structure of its N-terminal domain, Obg might interact with other structured RNAs to coordinate translation with other cellular processes .

• Metabolic sensing beyond GTP/GDP: Obg's role as a GTP/GDP sensor is established, but it might also respond to other metabolic signals. For instance, potential interactions with other nucleotides or metabolites could link translation to broader aspects of cellular metabolism .

• Heterogeneity of ribosome populations: How Obg might differentially interact with specialized ribosome subpopulations remains unknown. Recent research has revealed heterogeneity in ribosome composition and modification states; Obg might preferentially target specific ribosome variants to coordinate specialized translation programs .

• Eukaryotic Obg homologs: While bacterial Obg proteins have been extensively studied, eukaryotic homologs like GTPBP5 in human mitochondria deserve further investigation. These proteins might serve as critical links between mitochondrial ribosome assembly, translation, and cellular energy status, potentially contributing to mitochondrial diseases when dysregulated .

How might systems biology approaches enhance our understanding of Obg networks?

Systems biology approaches offer powerful frameworks for understanding Obg's role within broader cellular networks:

• Multi-omics integration: Combining transcriptomics, proteomics, and metabolomics data from wild-type versus Obg-depleted or mutant cells could reveal the global impact of Obg activity. This integrated approach would identify both direct and indirect consequences of Obg function, highlighting its role as a master regulator connecting translation to other cellular processes .

• Network analysis: Constructing interaction networks centered on Obg could identify functional modules and regulatory hubs that coordinate with Obg activity. This approach might reveal unexpected connections between Obg and other cellular pathways, providing a more comprehensive understanding of its regulatory role .

• Mathematical modeling: Developing quantitative models of Obg activity based on its biochemical properties could predict how changes in cellular conditions affect its function. These models could integrate parameters like GTP/GDP ratio, ribosome availability, and stress signals to predict how Obg coordinates cellular responses to changing environments .

• Comparative genomics: Systematic analysis of Obg proteins across diverse species could reveal evolutionary patterns in its structure and function. Identifying conserved versus variable features would highlight core functions while also revealing species-specific adaptations that might be relevant for targeted antimicrobial development .

• Synthetic biology applications: Engineered Obg variants with modified properties could serve as tunable regulators of bacterial physiology. By systematically altering Obg's response to specific signals, researchers could create bacterial strains with customized stress responses for biotechnological applications or gain fundamental insights into the design principles of cellular regulatory networks .