Recombinant Alkaliphilus metalliredigens GTPase Era (era)

What is Alkaliphilus metalliredigens GTPase Era and what are its primary functions?

Alkaliphilus metalliredigens GTPase Era is a 295-amino acid protein (UniProt No. A6TSJ8) from the metal-reducing bacterium Alkaliphilus metalliredigens strain QYMF. As a member of the highly conserved Era (E. coli Ras-like protein) family of GTPases, it plays critical roles in ribosome assembly, cell cycle regulation, and potentially in metal reduction pathways. The full amino acid sequence of this protein reveals conserved GTP-binding domains characteristic of bacterial GTPases .

Era proteins are essential in most bacteria, functioning as checkpoint regulators that coordinate cell division with ribosome assembly. They bind to the 30S ribosomal subunit in a GTP-dependent manner and likely contribute to quality control in ribosome biogenesis. In extremophiles like Alkaliphilus metalliredigens, Era may have additional specialized functions related to growth in alkaline, metal-rich environments.

What are the basic characteristics of Alkaliphilus metalliredigens as a source organism?

Alkaliphilus metalliredigens is a gram-positive, alkaliphilic, anaerobic bacterium with remarkable metal-reducing capabilities. It can use Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, or Cr(VI) as electron acceptors with yeast extract or lactate as electron donors. This bacterium demonstrates the following growth characteristics:

• pH range: 7.5 to 11.0 (optimal at pH 9.6)

• Sodium chloride tolerance: 0 to 80 g/l (optimal at 20 g/l)

• Temperature range: 4°C to 45°C (optimal at approximately 35°C)

• Borax tolerance: up to 1.5% (w/v)

The cells are straight rods that produce endospores, a feature that contributes to their survival in harsh environments. This organism is particularly interesting as it represents a novel metal-reducing bacterium that is distantly related to other commonly studied iron-reducing microorganisms .

How does the Recombinant Alkaliphilus metalliredigens GTPase Era differ from native Era protein?

The recombinant form of Alkaliphilus metalliredigens GTPase Era is expressed in E. coli expression systems and purified to >85% purity as determined by SDS-PAGE. While the amino acid sequence is identical to the native protein, the recombinant version may include additional tag sequences depending on the manufacturing process .

What are the recommended storage and handling conditions for Recombinant Alkaliphilus metalliredigens GTPase Era?

For optimal stability and activity retention, the following storage and handling guidelines should be followed:

• Store at -20°C for routine use, or at -80°C for extended storage

• Avoid repeated freezing and thawing cycles

• Working aliquots can be maintained at 4°C for up to one week

• The shelf life in liquid form is approximately 6 months at -20°C/-80°C

• The shelf life in lyophilized form extends to 12 months at -20°C/-80°C

When working with the protein, minimize exposure to conditions that could promote denaturation, such as extreme pH changes, high temperatures, or oxidizing agents.

What is the recommended protocol for reconstituting lyophilized GTPase Era?

To properly reconstitute lyophilized Recombinant Alkaliphilus metalliredigens GTPase Era:

• Briefly centrifuge the vial before opening to ensure the product is at the bottom

• Reconstitute in deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (50% is the standard recommendation)

• Create multiple small-volume aliquots to minimize freeze-thaw cycles

• Store reconstituted aliquots at -20°C/-80°C for long-term storage

This reconstitution protocol helps maintain protein stability and activity while reducing the risk of contamination or degradation during storage.

What buffer conditions are optimal for enzymatic activity assays with GTPase Era?

When designing enzyme activity assays for Alkaliphilus metalliredigens GTPase Era, consider the alkaliphilic nature of the source organism. The following buffer conditions are recommended:

Since GTPases require divalent cations (typically Mg²⁺) for catalytic activity, ensure that these ions are present in your assay buffer. Additionally, monitor pH carefully, as the enzyme may exhibit different kinetic properties at varying pH values, particularly given its origin from an alkaliphilic organism .

How can researchers investigate the role of GTPase Era in ribosome assembly in Alkaliphilus metalliredigens?

To study the role of GTPase Era in ribosome assembly, researchers can employ several complementary approaches:

• Co-immunoprecipitation studies: Using antibodies against Era to pull down associated ribosomal components, followed by mass spectrometry to identify interaction partners.

• Cryo-electron microscopy: To visualize the physical interaction between Era and the 30S ribosomal subunit at high resolution.

• Ribosome profiling: Comparing ribosome assembly patterns in wild-type cells versus those with depleted or mutated Era protein.

• In vitro reconstitution assays: Using purified components to reconstruct ribosome assembly with and without Era protein.

• Site-directed mutagenesis: Creating specific mutations in functional domains of Era to determine their impact on ribosome binding and assembly.

These methodologies can help elucidate how Era's GTPase activity coordinates with ribosome assembly, particularly in the context of an extremophile bacterium adapted to alkaline environments .

What approaches can be used to study potential links between GTPase Era and metal reduction in Alkaliphilus metalliredigens?

Investigating the potential role of GTPase Era in metal reduction processes requires multidisciplinary approaches:

• Metal binding assays: Using isothermal titration calorimetry or fluorescence spectroscopy to quantify binding affinities between purified Era and various metal ions.

• Metal reduction assays: Measuring the reduction of Fe(III), Co(III), or Cr(VI) in the presence of purified Era protein and appropriate electron donors.

• Structural studies: Crystallography or NMR studies of Era in the presence of different metals to identify potential binding sites.

• Genetic approaches: Creating Era knockdown or knockout strains to observe effects on metal reduction capabilities.

• Transcriptomic analysis: Comparing gene expression patterns between wild-type and Era-mutant strains during growth with different metal electron acceptors.

Given that Alkaliphilus metalliredigens is capable of reducing various metals including Fe(III)-citrate, Fe(III)-EDTA, Co(III)-EDTA, and Cr(VI), understanding Era's potential involvement could provide insights into novel metal reduction mechanisms .

How does GTPase Era from alkaliphilic bacteria differ structurally and functionally from Era in neutralophilic bacteria?

Understanding the adaptations of GTPase Era in alkaliphilic bacteria requires comparative analysis:

• Sequence comparison: Analysis of Era sequences from alkaliphilic versus neutralophilic bacteria reveals conservation of GTP-binding domains but variations in surface-exposed residues.

• Electrostatic surface analysis: Alkaliphilic proteins often show increased negative surface charge to maintain stability at high pH. Computational analysis of Era's electrostatic surface could reveal adaptations to alkaline environments.

• pH-dependent activity profiling: Comparing the GTPase activity of Era from Alkaliphilus metalliredigens with Era from neutralophilic bacteria across a pH gradient.

• Structural studies: X-ray crystallography or cryo-EM studies comparing the three-dimensional structures of Era proteins from different pH-adapted bacteria.

• Thermal stability analysis: Differential scanning calorimetry to compare the stability of Era proteins from various sources under different pH conditions.

These approaches can help identify specific adaptations that allow Era to function efficiently in the alkaline environment of Alkaliphilus metalliredigens .

What are the key considerations for designing experiments to study Era's interaction with the ribosome?

When investigating Era-ribosome interactions in Alkaliphilus metalliredigens, researchers should consider:

• Nucleotide state: Era's interaction with ribosomes is GTP-dependent. Experiments should compare binding in the presence of GTP, GDP, and non-hydrolyzable GTP analogs.

• Buffer conditions: Use buffers that mimic the alkaline intracellular environment of Alkaliphilus metalliredigens (pH 9.0-9.6).

• Salt concentration: Include appropriate concentrations of salt (optimally around 20 g/l NaCl) to reflect the organism's natural environment.

• Temperature: Conduct experiments at temperatures within the organism's growth range, ideally around 35°C.

• Control proteins: Include other GTPases or mutated versions of Era as controls to establish specificity of observed interactions.

• Ribosome preparation: Ensure that ribosomal preparations are free from contaminating proteins that might influence results.

These considerations will help ensure that experimental conditions appropriately reflect the physiological environment in which Era functions .

What methodological approaches can resolve contradictory results in GTPase Era activity assays?

When faced with contradictory results in GTPase Era activity assays, consider the following methodological approaches:

• Standardize protein preparation: Ensure consistent purification methods to avoid variability in protein quality or conformation.

• Validate enzyme activity: Use multiple assay methods (e.g., colorimetric phosphate release, HPLC-based nucleotide analysis) to cross-validate activity measurements.

• Control for metal ion effects: Carefully control the concentration and type of divalent cations, as these significantly impact GTPase activity.

• pH considerations: Given the alkaliphilic nature of the source organism, ensure precise pH control and consider that optimal pH for the recombinant protein may differ from expectations.

• Temperature effects: Conduct assays at multiple temperatures within the organism's growth range to identify temperature-dependent activity patterns.

• Time-course analysis: Perform detailed kinetic analysis rather than single-timepoint measurements to better understand reaction dynamics.

• Check for inhibitors or activators: Test for the presence of co-purified factors that might influence activity.

These approaches help identify sources of variability and establish reproducible conditions for accurate activity measurements .

What bioinformatic approaches are most valuable for studying GTPase Era structure-function relationships?

Advanced bioinformatic analyses can provide significant insights into Era's structure and function:

• Multiple sequence alignment: Comparing Era sequences across diverse bacterial species to identify conserved functional domains and species-specific variations.

• Homology modeling: Using crystal structures of Era from other bacteria as templates to predict the three-dimensional structure of Alkaliphilus metalliredigens Era.

• Molecular dynamics simulations: Simulating Era's behavior under different conditions (pH, temperature, salt concentration) to predict stability and conformational changes.

• Evolutionary trace analysis: Identifying functionally important residues by mapping conservation patterns onto structural models.

• Protein-protein interaction prediction: Computational prediction of potential interaction partners based on surface compatibility and known interactors in other species.

• Function prediction from structure: Using structural features to predict potential enzymatic activities beyond classical GTPase function.

These approaches can guide experimental design and help interpret experimental results in the broader context of GTPase Era evolution and adaptation .

How can researchers analyze kinetic data from GTPase Era enzymatic assays?

Proper analysis of kinetic data requires several analytical approaches:

• Michaelis-Menten kinetics: Determine Km, Vmax, and kcat values under various conditions to characterize the enzyme's catalytic efficiency.

• Arrhenius plot analysis: Examine the temperature dependence of reaction rates to determine activation energy.

• pH-rate profiles: Construct pH-activity curves to identify ionizable groups important for catalysis.

• Initial velocity studies: Analyze the initial linear portion of progress curves to avoid complications from product inhibition or substrate depletion.

• Global data fitting: Use software for global fitting of multiple datasets to complex kinetic models.

• Statistical validation: Apply appropriate statistical tests to determine the significance of observed kinetic differences under varying conditions.

For comparing Era activity across different conditions relevant to Alkaliphilus metalliredigens, consider using the following table format to organize results:

This structured approach allows for systematic comparison of enzymatic parameters across conditions relevant to the organism's environmental adaptations .

What are common challenges in expressing and purifying recombinant Alkaliphilus metalliredigens GTPase Era?

Researchers working with this protein may encounter several challenges:

• Protein solubility issues: Alkaliphile proteins often have unique solubility properties. Consider using:

  • Lower induction temperatures (16-20°C)

  • Solubility-enhancing fusion tags (SUMO, MBP, or TRX)

  • Expression in specialized E. coli strains designed for difficult proteins

  • Alkaline buffer conditions during lysis and purification

• Maintaining enzyme activity: GTPases can lose activity during purification due to:

  • Loss of bound nucleotides

  • Oxidation of critical cysteine residues

  • Improper folding during expression

  Address these by including GTP or non-hydrolyzable GTP analogs during purification, adding reducing agents, and optimizing buffer pH.

• Low expression yields: Improve yields by:

  • Codon optimization for E. coli

  • Testing multiple expression vectors with different promoters

  • Optimizing cell density at induction

  • Extending expression time at lower temperatures

• Protein aggregation: Prevent aggregation by:

  • Including appropriate detergents or stabilizers

  • Using buffers at pH 9.0-9.6 to mimic native conditions

  • Adding glycerol (5-10%) to stabilize protein structure

  • Incorporating salt at concentrations mimicking the organism's natural environment

How can researchers optimize GTPase activity assays for Alkaliphilus metalliredigens Era?

To develop robust activity assays:

• Select appropriate assay methods:

  • Malachite green phosphate release assay for high-throughput screening

  • HPLC analysis of GTP/GDP conversion for detailed kinetic studies

  • Real-time assays using fluorescent GTP analogs for continuous monitoring

• Buffer optimization:

  • Test buffers across pH range 7.5-11.0 with optimal around pH 9.6

  • Include appropriate salt concentration (optimal around 20 g/l NaCl)

  • Ensure sufficient Mg²⁺ concentration (typically 5-10 mM)

  • Add stabilizing agents (glycerol, BSA) to prevent protein adsorption to surfaces

• Temperature considerations:

  • Conduct assays at temperatures reflecting the organism's growth range (4-45°C)

  • Pre-incubate all components to the target temperature before initiating reactions

  • Monitor temperature stability throughout the assay duration

• Controls and validations:

  • Include no-enzyme controls to account for spontaneous GTP hydrolysis

  • Use known GTPases with well-characterized activity as positive controls

  • Verify that activity is proportional to enzyme concentration

  • Confirm linearity of the assay over the time course used

How can GTPase Era research contribute to understanding extremophile adaptation mechanisms?

Research on Alkaliphilus metalliredigens GTPase Era can provide valuable insights into extremophile adaptation:

• Molecular basis of alkaline adaptation: By comparing Era's structure and function with counterparts from neutralophilic bacteria, researchers can identify specific adaptations that enable protein stability and function at high pH.

• Metal tolerance mechanisms: Investigating Era's potential role in metal reduction pathways could reveal novel mechanisms for metal tolerance and utilization in extreme environments.

• Stress response coordination: Era's role in coordinating ribosome assembly with cell division may have evolved unique features in extremophiles to respond to environmental stressors.

• Evolution of essential cellular machinery: Comparing Era across diverse extremophiles can illuminate how essential cellular processes have been modified during adaptation to extreme environments.

• Protein engineering principles: Identifying the structural features that confer stability under extreme conditions can inform protein engineering efforts for biotechnological applications .

What are promising research directions for exploring GTPase Era's role in metal reduction processes?

Future research on Era's potential role in metal reduction could focus on:

• Direct versus indirect involvement: Determining whether Era directly participates in metal reduction or indirectly regulates other proteins involved in these pathways.

• Redox-sensitive regulation: Investigating whether Era's GTPase activity or ribosome binding is modulated by redox state or metal availability.

• Metal-dependent localization: Exploring whether Era's subcellular localization changes in response to different metals or redox conditions.

• Regulatory networks: Mapping the gene regulatory networks connecting Era to known metal reduction pathways using transcriptomics and proteomics approaches.

• Comparative studies: Contrasting Era's functions in Alkaliphilus metalliredigens with those in non-metal-reducing bacteria to identify specializations related to metal reduction.

Understanding these aspects could provide insights into the evolutionary adaptations that allow Alkaliphilus metalliredigens to thrive in metal-rich alkaline environments, with potential applications in bioremediation of metal-contaminated sites .