Recombinant Deinococcus radiodurans Putative zinc metalloprotease DR_1507 (DR_1507)

What is DR_1507 and what is its basic structure?

DR_1507 is a putative zinc metalloprotease from the extremophile bacterium Deinococcus radiodurans. The full-length protein consists of 377 amino acids and is typically studied as a recombinant protein with an N-terminal His tag when expressed in E. coli expression systems . The protein's amino acid sequence (MNVLQGMAAALTPLGLLWTAIIFGVSVFLHELAHYGLARAQGVRVNSFSVGMGPVLFKKLWRGTEWRVSLLPIGGYVEIDGMAPVEDADGQWRLPTRGFAALPAWGKIAVLLAGPLTNLLLTLGLMTVSFTSQGIPALDRARIESVETGSRAQALGLRAGDVITAIDGQDIPETRRVGGQEAAGYEGVRDALAQAGRHTFTVERAEQGQPVQTRQVAFDWQPTVNGQRQLLGIRYGPDVRQVGVGQAFVTSVDTTVRAVPQLVGAFTGLFKKFFTLDISQDQNVSGPIGTAEVISRAAALSPWALVQVATLLNLSLAFFNLIPIPGLDGGRILLVLVSALRGRPLSFQQEQAINLGGFAFVMLLTLFVVVRDVSRFF) contains structural motifs characteristic of metalloproteases .

Why is Deinococcus radiodurans significant as a research organism?

Deinococcus radiodurans is an extremophile known for its exceptional tolerance to ionizing radiation (IR) and other forms of stress. This bacterium constitutively maintains a highly condensed nucleoid structure, which may contribute to its remarkable stress resistance properties . Recent research has focused on understanding the relationship between nucleoid organization and the organism's ability to withstand extreme conditions, making D. radiodurans an important model for studying cellular stress response mechanisms .

What is the optimal reconstitution protocol for recombinant DR_1507?

For optimal reconstitution of lyophilized DR_1507, first centrifuge the vial briefly to ensure all content is at the bottom. Reconstitute the protein in deionized sterile water to a concentration of 0.1-1.0 mg/mL. For long-term storage, add glycerol to a final concentration of 5-50% (with 50% being recommended by suppliers) and aliquot before storing at -20°C/-80°C to avoid repeated freeze-thaw cycles which may compromise protein integrity . Working aliquots can be stored at 4°C for up to one week .

What is the role of zinc in metalloproteases like DR_1507?

Zinc plays a critical role in the structure and function of metalloproteases. Based on studies of similar zinc metalloproteases, the metal ion is typically coordinated by specific amino acid residues, often including a conserved HEXXH motif where the two histidine residues participate in zinc coordination . The zinc ion is essential for catalytic activity, as it helps position a water molecule for nucleophilic attack during peptide bond hydrolysis. Removal of zinc (such as by chelation with EDTA) typically results in loss of enzymatic activity, while reconstitution with zinc can restore function .

How can I determine the substrate specificity of DR_1507?

Determining substrate specificity of DR_1507 requires a systematic approach:

• Initial substrate screening: Test the protein against common metalloprotease substrates including:

  • Casein and gelatin (via zymography)

  • Synthetic FRET (Fluorescence Resonance Energy Transfer) peptides

  • Physiologically relevant proteins (fibronectin, fibrinogen, etc.)

• Activity confirmation: Conduct time-course and concentration-dependent assays with identified substrates in the presence of zinc (0.5 mM ZnCl₂) as observed with similar metalloproteases .

• Cleavage site identification: For proteins showing degradation, perform:

  • SDS-PAGE analysis to identify fragment patterns

  • N-terminal sequencing of generated fragments using Edman degradation

  • Mass spectrometry to precisely map cleavage sites

• Metal dependency verification: Compare activity in the presence of different divalent cations (Zn²⁺, Cu²⁺, Ni²⁺) and chelating agents (EDTA) to confirm zinc-dependency .

Based on studies of similar bacterial zinc metalloproteases, potential substrates might include extracellular matrix proteins like fibronectin and fibrinogen, which have been shown to be cleaved by other bacterial zinc-dependent proteases in a time and concentration-dependent manner .

What mutagenesis approaches are most informative for studying DR_1507 function?

Site-directed mutagenesis focusing on the catalytic domains of DR_1507 provides crucial insights into its function:

• Target residues in putative zinc-binding motifs:

  • Focus on the HEXXH motif if present (particularly the glutamate and histidine residues)

  • Based on studies of similar metalloproteases, mutations such as E→A and H→A in this motif can differentiate between residues involved in zinc coordination versus catalytic activity

• Experimental validation of mutants:

  • Protein stability assessment using differential scanning fluorimetry (DSF) to ensure mutations don't compromise protein folding

  • Zinc-binding capacity evaluation through thermal shift assays in the presence of zinc

  • NMR analysis to confirm metal binding properties of wild-type versus mutant proteins

• Activity comparisons:

  • Conduct parallel activity assays of wild-type and mutant proteins against identified substrates

  • Quantify differences in catalytic efficiency (kcat/KM) to determine the contribution of specific residues

This approach has successfully identified functional residues in other bacterial metalloproteases, where mutations in the HEXXH motif revealed differential effects on metal binding versus catalytic function .

How does ionizing radiation affect the expression and activity of DR_1507 in D. radiodurans?

Investigating the relationship between ionizing radiation (IR) exposure and DR_1507 requires a multi-faceted approach:

• Expression analysis:

  • Expose D. radiodurans cultures to varying doses of IR (typically 1-15 kGy)

  • Measure DR_1507 transcript levels using qRT-PCR at different time points during recovery

  • Quantify protein levels using Western blotting with anti-DR_1507 antibodies

• Correlation with cellular morphology:

  • Use confocal microscopy with fluorescent staining (Nile Red for cell membrane, Syto 9 Green for DNA) to track changes in nucleoid condensation following IR exposure

  • Apply quantitative image analysis to correlate DR_1507 expression with nucleoid organization changes

• Functional studies:

  • Compare proteolytic activity of DR_1507 isolated from irradiated versus non-irradiated cells

  • Investigate potential post-translational modifications induced by radiation exposure

Recent studies on D. radiodurans have shown that IR exposure results in complex, dose-dependent changes in nucleoid condensation during recovery . As a putative metalloprotease, DR_1507 might play a role in protein turnover during stress response, potentially contributing to the organism's remarkable radiation resistance.

What are the most effective methods for assessing zinc binding to DR_1507?

Multiple complementary techniques provide comprehensive assessment of zinc binding to DR_1507:

For DR_1507, a recommended approach would be:

• Initial screening using DSF to determine if zinc and other divalent cations (Cu²⁺, Ni²⁺) stabilize the protein structure

• Confirmation of binding using ICP-MS to quantify zinc:protein ratios

• Detailed characterization using NMR with ¹⁵N-labeled protein to identify specific residues involved in zinc coordination

This approach has been successfully applied to other metalloproteases, where zinc binding resulted in measurable increases in melting temperature and characteristic changes in NMR spectra .

How should I design experiments to compare DR_1507 with other bacterial zinc metalloproteases?

A comprehensive comparative analysis should include:

• Sequence and structural alignment:

  • Perform multiple sequence alignment of DR_1507 with characterized bacterial zinc metalloproteases

  • Focus on conservation of zinc-binding motifs (HEXXH) and catalytic residues

  • Generate homology models if crystal structures are unavailable

• Parallel biochemical characterization:

  • Express and purify DR_1507 alongside reference metalloproteases (e.g., Zmp1 from C. difficile )

  • Standardize buffer conditions (Tris-based buffers with controlled pH ~8.0)

  • Compare metal binding preferences using DSF and ICP-MS across multiple divalent cations

• Substrate profiling:

  • Test activity against a panel of substrates including casein, gelatin, fibronectin, and fibrinogen

  • For active substrates, perform side-by-side comparison of:

    • Time-dependent cleavage patterns (0-24h)

    • Concentration-dependent activity

    • Metal ion dependency

• Inhibitor sensitivity:

  • Evaluate response to common metalloprotease inhibitors (EDTA, 1,10-phenanthroline)

  • Test with specific zinc protease inhibitors to establish inhibition profiles

This approach will highlight functional similarities and differences between DR_1507 and other characterized bacterial zinc metalloproteases, potentially revealing unique features related to D. radiodurans' extreme stress tolerance .

What controls are essential when studying the enzymatic activity of DR_1507?

Critical controls for DR_1507 enzymatic activity studies include:

• Negative controls:

  • Substrate-only incubation to monitor spontaneous degradation

  • Heat-inactivated DR_1507 (boiled for 10 minutes) to confirm enzymatic nature of activity

  • Metal-free conditions (protein treated with EDTA) to verify metal dependency

• Positive controls:

  • Well-characterized zinc metalloprotease with known activity

  • Activity assays in optimal conditions (buffer, pH, temperature)

• Specificity controls:

  • DR_1507 mutants lacking critical catalytic residues (E→A, H→A in HEXXH motif)

  • Assays with different divalent cations (Zn²⁺, Cu²⁺, Ni²⁺) to establish metal specificity

  • Inclusion of specific metalloprotease inhibitors at varying concentrations

• Technical controls:

  • Freshly prepared versus stored enzyme preparations to account for potential activity loss

  • Multiple protein batches to ensure reproducibility

  • Range of substrate:enzyme ratios to establish linearity of reaction

Implementation of these controls will ensure that any observed activity can be reliably attributed to the zinc-dependent proteolytic activity of DR_1507, rather than contaminants or artifacts .

How can I establish an effective expression and purification system for DR_1507?

A systematic approach to DR_1507 expression and purification includes:

• Expression system optimization:

  • E. coli is the established system for DR_1507 expression

  • Compare different E. coli strains (BL21(DE3), Rosetta, Origami) for optimal expression

  • Test induction conditions: IPTG concentration (0.1-1.0 mM), temperature (16-37°C), duration (4-24h)

• Construct design considerations:

  • N-terminal His-tag has been successfully used for DR_1507

  • Consider testing alternative tags (GST, MBP) if solubility issues arise

  • Include a precision protease cleavage site for tag removal if needed for functional studies

• Purification strategy:

  • Initial capture: Ni-NTA affinity chromatography for His-tagged protein

  • Secondary purification: Size exclusion chromatography to ensure homogeneity

  • Verification of purity: SDS-PAGE with >90% purity target

• Buffer optimization:

  • Tris/PBS-based buffers (pH 8.0) with potential addition of:

    • Zinc supplementation (0.1-0.5 mM ZnCl₂) to maintain metallation state

    • Reducing agents (DTT or β-mercaptoethanol) to prevent oxidation

    • Glycerol (5-10%) to enhance stability

• Storage considerations:

  • Lyophilization with 6% trehalose for long-term storage

  • Aliquoting in 50% glycerol for frozen storage (-20°C/-80°C)

  • Stability testing at 4°C for working solutions (up to one week)

Following this pipeline has yielded functional recombinant DR_1507 with greater than 90% purity suitable for biochemical and structural studies .

What imaging techniques are most suitable for studying DR_1507 in the context of D. radiodurans nucleoid organization?

Optimal imaging of DR_1507 in relation to nucleoid organization requires:

• Sample preparation:

  • Culture D. radiodurans under standard conditions or following stress exposure (e.g., ionizing radiation)

  • Fix cells with paraformaldehyde (2-4%) to preserve structure

  • Dual staining with:

    • Nile Red for cell membrane visualization

    • Syto 9 Green for nucleoid DNA visualization

    • Immunofluorescence with anti-DR_1507 antibodies

• Imaging platforms:

  • Laser-scanning confocal microscopy for high-resolution 3D imaging

  • Super-resolution techniques (STORM, PALM) for nanoscale localization

  • Time-lapse microscopy for dynamic processes

• Quantitative analysis:

  • Digital reconstruction of cells from confocal images

  • Measurement of geometric parameters:

    • Cell area and shape

    • Nucleoid area and condensation state

    • DR_1507 localization relative to nucleoid

  • Statistical analysis of morphological changes across populations

This approach allows correlation between DR_1507 expression/localization and the characteristic nucleoid condensation observed in D. radiodurans, particularly following stress exposure .

How can I quantitatively assess changes in DR_1507 activity under different stress conditions?

A quantitative framework for measuring DR_1507 activity changes includes:

This methodology provides a comprehensive view of how DR_1507 activity responds to and potentially contributes to the remarkable stress resistance of D. radiodurans.

What bioinformatic approaches can predict potential substrates and interaction partners of DR_1507?

Computational prediction of DR_1507 substrates and interactors involves:

• Sequence-based analysis:

  • Motif identification using MEME, GLAM2 to identify potential cleavage sites

  • Comparison with known bacterial zinc metalloprotease substrate preferences

  • Scanning of D. radiodurans proteome for proteins containing predicted target motifs

• Structural modeling and docking:

  • Generate homology models of DR_1507 based on related metalloproteases

  • Molecular docking of potential substrates to assess binding compatibility

  • Molecular dynamics simulations to evaluate stability of enzyme-substrate complexes

• Network analysis:

  • Gene neighborhood analysis to identify functionally related proteins

  • Co-expression analysis from transcriptomic data, particularly under stress conditions

  • Protein-protein interaction prediction using STRING, PSICQUIC

• Experimental validation strategies:

  • Pull-down assays with recombinant DR_1507 followed by mass spectrometry

  • Bacterial two-hybrid screening

  • In vitro cleavage assays with candidate substrates

This integrated computational and experimental pipeline can generate testable hypotheses about DR_1507's biological function in D. radiodurans, particularly in relation to its extreme stress resistance.

How might DR_1507 contribute to D. radiodurans' extreme radiation resistance?

Based on current understanding of zinc metalloproteases and D. radiodurans biology, several hypotheses emerge:

• Protein quality control:

  • DR_1507 may participate in degrading damaged proteins following radiation exposure

  • This function would complement the efficient DNA repair mechanisms known in D. radiodurans

• Cell envelope maintenance:

  • As a putative membrane-associated metalloprotease, DR_1507 might modulate cell envelope proteins

  • This could contribute to maintaining membrane integrity during stress

• Stress signaling:

  • Proteolytic processing of specific substrates might activate or inactivate stress response pathways

  • This could coordinate cellular responses to radiation damage

• Nucleoid organization:

  • DR_1507 might process nucleoid-associated proteins, potentially contributing to the characteristic condensed nucleoid structure observed in D. radiodurans

  • The highly organized nucleoid may protect DNA from radiation damage

Research approaches to test these hypotheses include:

• Generation of DR_1507 knockout strains and assessment of radiation sensitivity

• Identification of DR_1507 substrates before and after radiation exposure

• Correlation of DR_1507 activity with nucleoid condensation dynamics

What comparative studies between DR_1507 and metalloproteases from non-extremophiles would be most informative?

Strategic comparative studies should focus on:

• Structural stability comparisons:

  • Thermal stability analysis (DSF, circular dichroism) across temperature ranges

  • Resistance to denaturation under extreme conditions (high salt, pH extremes, organic solvents)

  • Metal-binding affinity and specificity across diverse environmental conditions

• Functional conservation assessment:

  • Substrate specificity comparison with metalloproteases from non-extremophiles

  • Activity retention under stress conditions that inactivate non-extremophile enzymes

  • Ability to complement function in heterologous systems

• Evolutionary analysis:

  • Identification of unique sequence features through multiple sequence alignment

  • Examination of selective pressure on DR_1507 versus homologs (dN/dS analysis)

  • Reconstruction of evolutionary history to identify adaptations to extreme environments

• Domain swapping experiments:

  • Creation of chimeric proteins with domains from extremophile and non-extremophile metalloproteases

  • Assessment of which domains confer extremophile-like properties

These approaches would reveal whether DR_1507 has evolved specific adaptations that contribute to D. radiodurans' extremophile lifestyle, potentially identifying novel mechanisms of protein stability and function under extreme conditions.