Recombinant Sulfolobus solfataricus DNA protection during starvation protein (dps)

What is Sulfolobus solfataricus DNA Protection During Starvation Protein (Dps)?

SsDps is an archaeal antioxidant protein of approximately 22 kDa isolated from the hyperthermophilic acidophile Sulfolobus solfataricus. Despite sharing limited sequence similarity with proteins of known function, it belongs to a monophyletic cluster within the ferritin-like diiron-carboxylate protein superfamily . The protein functions primarily to protect nucleic acids against oxidative damage through two mechanisms: physically shielding DNA and consuming constituents involved in Fenton chemistry. SsDps efficiently uses H₂O₂ to oxidize Fe(II) to Fe(III) and stores the resulting iron oxide as a mineral core on the interior surface of its protein cage .

What is the structural organization of SsDps?

SsDps self-assembles into a hollow dodecameric protein cage with tetrahedral symmetry. The assembled structure has an outer shell diameter of approximately 10 nm and an interior diameter of approximately 5 nm . This quaternary structure is structurally related to the class of DNA-binding proteins from starved cells (Dps) found in bacteria. The protein contains an N-terminal extension similar to that found in DPS molecules, which is thought to mediate interactions with DNA . X-ray crystallography has revealed that SsDps contains a Bacterioferritin-like dimetal binding site within its DPS-like dodecameric assembly .

How does SsDps function as an antioxidant?

SsDps exhibits antioxidant activity through multiple mechanisms:

• Iron Sequestration: The protein efficiently catalyzes the oxidation of Fe(II) to Fe(III) using H₂O₂ as an oxidant, storing the resulting iron oxide as a mineral core inside its hollow structure .

• H₂O₂ Consumption: By utilizing H₂O₂ in the oxidation of iron, SsDps reduces the concentration of this reactive oxygen species, preventing hydroxyl radical formation via Fenton chemistry .

• DNA Protection: SsDps is believed to physically associate with DNA, providing direct protection against oxidative damage. This association, combined with its iron mineralization activity, represents a multifunctional approach to cellular protection .

What are the optimal storage conditions for recombinant SsDps?

For optimal stability, recombinant SsDps should be stored at -20°C, or at -20°C/-80°C for extended storage periods . For working aliquots, storage at 4°C for up to one week is recommended. Repeated freezing and thawing should be avoided to maintain protein integrity. For reconstitution, the protein should be dissolved in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of 5-50% glycerol (final concentration) for long-term storage . The shelf life is approximately 6 months for liquid form at -20°C/-80°C and 12 months for lyophilized form at the same temperature ranges.

How is gene expression of ssdps regulated in S. solfataricus?

The ssdps gene expression in S. solfataricus is highly responsive to specific environmental stressors. Northern blot analysis has demonstrated that:

• Oxidative Stress: The transcript is up-regulated in a nonlinear fashion in response to H₂O₂ exposure, with a dramatic increase observed at 30 μM H₂O₂. Western blots indicate a more linear accumulation of the SsDps protein with increasing H₂O₂ concentrations .

• Iron Availability: Transcription is significantly increased in iron-depleted media, suggesting a role in iron homeostasis .

• Stress Specificity: Interestingly, ssdps transcription is not elevated in response to other stressors including UV irradiation, alternative carbon sources, virus infection, heat shock (90°C), or cold shock (60°C). This indicates that SsDps is specifically involved in oxidative stress response rather than being part of a general stress response system .

What methods can be used to purify SsDps from different expression systems?

Purification methodologies differ depending on whether SsDps is being isolated from native S. solfataricus or from recombinant expression systems:

• From S. solfataricus cultures:

  • Induce expression by supplementing culture media with 30 μM H₂O₂

  • Expected yield: approximately 0.3 mg from a 1-liter culture

  • Standard purification protocols yield negligible amounts from unstressed cultures

• From recombinant E. coli:

  • Expression yields approximately 40 mg from a 1-liter culture

  • Leverage the protein's thermal stability by incorporating a heat treatment step (10 minutes at 65°C) to denature most E. coli proteins

  • Continue with standard chromatographic techniques for final purification

The dramatic difference in yield (>100-fold) makes recombinant expression the preferred method for obtaining sufficient quantities for structural and biochemical studies.

How do mutations in conserved DNA and metal-binding residues affect SsDps function?

Studies of SisPerR, another redox-sensing transcription factor in Sulfolobus islandicus with functional similarities to SsDps, provide insights into the importance of conserved residues:

• DNA-binding residues: The threonine residue at position 53 (analogous to threonine 41 in Clostridioides difficile PerR) is highly conserved in both bacterial and archaeal PerR proteins. Mutation of this residue to alanine (T53A) abolishes the H₂O₂-sensing ability of the protein .

• Functional consequences: Complementation of sisperR deletion with PerR(T53A) results in a strain whose growth under oxidative stress (70 μM H₂O₂) is similar to that of the deletion strain, confirming the critical nature of this residue .

• Phenotypic effects: While wild-type overexpression strains exhibit growth retardation, cell enlargement, and aggregation (signs of DNA damage and cell cycle regulation stress), strains expressing the T53A mutant grow normally under standard conditions and show only slight growth retardation with H₂O₂ treatment .

These findings suggest that conserved residues in redox-sensing proteins of Sulfolobales are essential for their function in oxidative stress responses and likely play similar roles in SsDps.

What experimental approaches can be used to study SsDps interaction with DNA?

While specific methods for studying SsDps-DNA interactions were not detailed in the search results, standard approaches for investigating Dps-DNA interactions include:

• Electrophoretic Mobility Shift Assays (EMSA): To detect direct binding between purified SsDps and DNA fragments.

• DNA protection assays: Measuring the ability of SsDps to prevent DNA damage under oxidative stress conditions, typically using plasmid DNA and monitoring the transition between supercoiled, nicked, and linear forms.

• Fluorescence microscopy: Using fluorescently labeled SsDps and DNA to visualize co-localization in vitro or in vivo.

• Co-immunoprecipitation: To detect SsDps-DNA complexes from cellular extracts using antibodies against SsDps.

The N-terminal extension of SsDps, similar to that found in bacterial Dps proteins, is thought to mediate DNA interactions . This region would be a primary target for mutation studies to confirm its role in DNA binding.

How can the iron storage capacity of SsDps be assessed in vitro?

To evaluate the iron storage capacity of SsDps, researchers can employ several complementary techniques:

• Ferroxidase activity assay: Monitoring the rate of Fe(II) oxidation in the presence of H₂O₂ by measuring the disappearance of Fe(II) using colorimetric reagents such as ferrozine.

• Iron loading quantification: Determining the maximum iron:protein ratio by incubating SsDps with excess Fe(II) under oxidizing conditions, followed by removal of unbound iron and quantification of bound iron using atomic absorption spectroscopy or inductively coupled plasma mass spectrometry.

• Mineral core characterization: Analyzing the formed mineral core using transmission electron microscopy with or without energy-dispersive X-ray spectroscopy to determine the structure and composition of the stored iron minerals.

• Kinetic studies: Measuring the rate of iron oxidation as a function of H₂O₂ concentration to determine the preference of SsDps for H₂O₂ as an oxidant compared to O₂, which is a characteristic feature of Dps proteins.

How does SsDps differ from bacterial Dps proteins in terms of structure and function?

While SsDps shares fundamental similarities with bacterial Dps proteins, several key differences have been identified:

• Sequence divergence: SsDps shares limited sequence similarity with proteins of known function but forms a distinct monophyletic cluster with other archaeal Dps-like proteins within the ferritin superfamily .

• Structural features: SsDps contains a Bacterioferritin-like dimetal binding site within its DPS-like dodecameric assembly, representing a unique structural feature .

• Oxidative stress response: SsDps is specifically induced by H₂O₂ and iron depletion but not by other stressors, whereas some bacterial Dps proteins respond to a broader range of stress conditions .

• Environmental adaptation: SsDps functions in a hyperthermophilic, acidophilic environment (70-90°C, pH 3.3), requiring adaptations not present in mesophilic bacterial Dps proteins .

• Oxidative stress tolerance: Sulfolobus solfataricus cells have a significantly higher basal intracellular ROS level (about 5 × 10³) compared to aerobic mesophiles like E. coli (<10²), yet are more sensitive to exogenous H₂O₂ (lethal at 200 μM vs. 6 mM for E. coli) .

What insights does SsDps provide about the evolution of oxidative stress responses?

SsDps offers valuable evolutionary insights into antioxidant mechanisms:

• Ancient origins: As Sulfolobus solfataricus is a deeply rooted organism in the tree of life, SsDps likely represents an ancient mechanism for managing oxidative stress .

• Selective pressure: The evolution of oxygenic photosynthesis created one of the greatest selective pressures on primordial life. SsDps exemplifies how early life forms developed strategies to mitigate the toxicity of reactive oxygen species .

• Conserved mechanisms: Despite significant sequence divergence, the structural and functional conservation between archaeal and bacterial Dps proteins suggests that the ferritin-like protein cage architecture represents a fundamental solution to the challenge of oxidative stress .

• Specialized adaptations: The high basal ROS levels in S. solfataricus and its hyperthermophilic lifestyle indicate specialized adaptations to manage oxidative stress in extreme environments, potentially representing unique evolutionary solutions .

What are the challenges in working with hyperthermophilic archaeal proteins like SsDps?

Working with hyperthermophilic archaeal proteins presents several unique challenges:

• Growth conditions: Culturing Sulfolobus solfataricus requires specialized conditions including high temperature (75°C), acidic pH (3.3), and specific media components .

• Low yield from native sources: Native purification from S. solfataricus yields only about 0.3 mg/L when induced with H₂O₂, making recombinant expression preferable for obtaining research quantities .

• Protein stability considerations: While SsDps is extremely stable at high temperatures, attention must be paid to maintaining its stability during purification and analysis at lower temperatures typically used in laboratory settings.

• Reconstitution concerns: Special attention must be given to proper reconstitution of the lyophilized protein, with recommendations to centrifuge vials before opening and to add glycerol for long-term storage .

• Functional analysis: Standard assays may need modification to account for the protein's optimal activity at high temperatures and acidic pH levels.

How can the thermal stability of SsDps be leveraged in experimental protocols?

The exceptional thermal stability of SsDps offers several advantages that can be exploited in laboratory procedures:

• Purification enhancement: A simple heat treatment (10 minutes at 65°C) can be used as an initial purification step when working with recombinant SsDps expressed in E. coli, as this denatures most host proteins while leaving SsDps intact .

• Long-term storage: The thermal stability contributes to extended shelf life (6 months for liquid form, 12 months for lyophilized form at -20°C/-80°C) .

• Assay flexibility: Activity assays can be performed across a wide temperature range, allowing for comparative studies of enzyme kinetics under different thermal conditions.

• Industrial applications: The thermostability makes SsDps a potential candidate for applications requiring stable protein scaffolds in high-temperature environments.

What analytical techniques are most suitable for characterizing SsDps assembly and iron loading?

Several analytical approaches are particularly valuable for studying SsDps:

• Structural characterization:

  • Transmission electron microscopy for direct visualization of protein cage assembly

  • Dynamic light scattering for measuring particle size distribution

  • Analytical ultracentrifugation for determination of assembly state

• Iron mineralization:

  • UV-visible spectroscopy to monitor iron core formation (absorbance at 350-400 nm)

  • Mössbauer spectroscopy for detailed characterization of the iron mineral phase

  • Atomic absorption spectroscopy or ICP-MS for precise quantification of iron content

• Functional analysis:

  • Ferroxidase activity assays with various oxidants (H₂O₂ vs. O₂)

  • DNA protection assays under oxidative stress conditions

  • ROS scavenging measurements using fluorescent probes like DCFH-DA