Recombinant Sulfolobus solfataricus Phosphoglycolate phosphatase 1 (SSO0094)

What is Sulfolobus solfataricus Phosphoglycolate phosphatase 1 (SSO0094)?

Sulfolobus solfataricus Phosphoglycolate phosphatase 1 (SSO0094) is an enzyme encoded by the SSO0094 gene from the hyperthermophilic archaeon Sulfolobus solfataricus strain ATCC 35092/DSM 1617/JCM 11322/P2. This enzyme belongs to the phosphatase family (EC 3.1.3.18) and catalyzes the hydrolysis of phosphoglycolate to glycolate and inorganic phosphate. The full-length protein consists of 228 amino acids and has a molecular weight typically falling in the range of 24-26 kDa, depending on any additional tags used in the recombinant form .

What are the optimal storage conditions for recombinant SSO0094?

For short-term storage (up to one week), recombinant Sulfolobus solfataricus Phosphoglycolate phosphatase 1 can be stored as working aliquots at 4°C. For long-term storage, the protein should be kept at -20°C, and for extended storage periods, conservation at -20°C or -80°C is recommended .

The protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL. To enhance stability during storage, it is advisable to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation for many commercial preparations). After reconstitution, the protein should be aliquoted to avoid repeated freeze-thaw cycles, which can compromise enzyme activity and structural integrity .

How can I confirm the purity and integrity of recombinant SSO0094?

To verify the purity and integrity of recombinant SSO0094, several analytical techniques can be employed:

• SDS-PAGE Analysis: Commercial preparations typically achieve >85% purity as assessed by SDS-PAGE . Researchers should run their protein samples alongside appropriate molecular weight markers to confirm the expected size.

• Western Blotting: If antibodies against SSO0094 or any epitope tags are available, Western blotting provides additional confirmation of identity.

• Mass Spectrometry: For more precise characterization, techniques such as MALDI-TOF or ESI-MS can verify the exact molecular weight and confirm post-translational modifications.

• Activity Assays: Functional assays measuring the phosphatase activity using appropriate substrates provide information about the catalytic integrity of the enzyme.

• Circular Dichroism: This technique can assess the secondary structure content, providing information about proper protein folding.

How does phosphorylation affect enzyme activity in archaeal phosphatases?

Phosphorylation can significantly impact the catalytic activity of archaeal phosphatases, as demonstrated in studies of related enzymes from Sulfolobus solfataricus. Research on a phosphohexomutase from this organism revealed that phosphorylation at specific serine residues within the substrate binding site can dramatically reduce enzymatic activity .

When Ser-309 in a phosphohexomutase from S. solfataricus was found to be phosphorylated, researchers investigated its impact through site-directed mutagenesis. By substituting Ser-309 with aspartic acid to mimic phosphorylation, they observed that the V(max) of the altered protein was only 4% that of the unmodified form. Even substitution with larger but uncharged amino acids like threonine decreased catalytic efficiency, though to a lesser extent (three- to fivefold reduction) .

By extrapolation, similar phosphorylation-based regulation may occur in SSO0094, suggesting a potential mechanism for regulating metabolic pathways in these extremophilic archaea. Researchers studying SSO0094 should consider investigating potential phosphorylation sites and their effects on catalytic activity.

What role might SSO0094 play during viral infection of Sulfolobus solfataricus?

During viral infection of Sulfolobus solfataricus, proteomics studies have shown significant changes in host protein concentrations. Although specific data for SSO0094 is not directly provided in the search results, research on Sulfolobus Turreted Icosahedral Virus (STIV) infection revealed that 71 host proteins changed concentration by nearly twofold, with 40 becoming more abundant and 31 less abundant .

These modulated proteins represented 30 different cell pathways and 14 clusters of orthologous groups. Importantly, post-translational modifications were a common feature of the affected proteins. Activity-based protein profiling (ABPP) on 2D-gels demonstrated caspase, hydrolase, and tyrosine phosphatase enzyme activity labeling at the protein isoform level .

As a phosphatase, SSO0094 may be involved in similar regulatory responses during viral infection. Phosphatases often play roles in signal transduction and metabolic regulation, suggesting that SSO0094 might participate in the archaeal host response to viral invasion, potentially through dephosphorylation of key substrates involved in antiviral defense mechanisms or metabolic adaptation.

How can small-angle scattering techniques be applied to study SSO0094 structural dynamics?

Small-angle scattering (SAS) techniques, including small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS), provide valuable tools for studying the structural dynamics of proteins like SSO0094 in solution. These techniques are particularly useful for investigating conformational changes that may occur during substrate binding or catalysis.

When applying SAS to study SSO0094, researchers should follow the updated reporting guidelines that include:

• Sample Preparation Details: Comprehensive documentation of protein concentration, buffer composition, and sample homogeneity is essential .

• Data Collection Parameters: Important parameters include the q-range, exposure time, temperature, and radiation damage assessment .

• Data Analysis and Modeling: The analysis should include:

  • Scattering profile (I(q) versus q on log-linear and log-log scales)

  • Dimensionless Kratky plots [(qRg)² I(q)/I(0) versus qRg] to assess the folding state

  • Pairwise distance distribution function [P(r) versus r] to determine maximum dimension

For studying substrate binding or conformational changes in SSO0094, contrast variation experiments might be particularly valuable. These experiments would require preparing samples with different D2O concentrations and potentially using deuterated substrates to enhance contrast .

The resulting structural models should be presented with comprehensive reporting of parameters such as Rg (radius of gyration), Dmax (maximum dimension), and molecular mass estimates from I(0) and Porod volume .

What are the optimal expression and purification strategies for obtaining active recombinant SSO0094?

Obtaining active recombinant SSO0094 requires careful consideration of expression systems and purification strategies. Based on established protocols for thermostable archaeal proteins:

Expression System Selection:

• E. coli: The most commonly used system for SSO0094 expression . BL21(DE3) or Rosetta strains are often preferred for archaeal proteins.

• Expression Temperature: Lower temperatures (16-25°C) after induction may enhance proper folding despite the thermophilic nature of the native protein.

• Induction Conditions: IPTG concentration (typically 0.1-1.0 mM) and induction time (4-16 hours) should be optimized for maximum yield of soluble protein.

Purification Strategy:

• Heat Treatment: Exploiting the thermostability of SSO0094 by heating the cell lysate (70-80°C for 10-30 minutes) to denature most E. coli proteins while leaving the archaeal enzyme intact.

• Immobilized Metal Affinity Chromatography (IMAC): If expressed with a His-tag, purification using Ni-NTA or similar matrices.

• Ion Exchange Chromatography: As a second purification step to remove contaminants with different charge properties.

• Size Exclusion Chromatography: Final polishing step to ensure homogeneity and remove aggregates.

Buffer Conditions:

• pH Range: Typically 6.0-8.0 for optimal stability during purification.

• Salt Concentration: 100-300 mM NaCl to maintain solubility.

• Reducing Agent: Addition of DTT or β-mercaptoethanol (1-5 mM) to prevent oxidation of cysteine residues.

Quality Control:
After purification, enzyme activity should be verified using appropriate phosphatase assays, and protein purity confirmed by SDS-PAGE (aiming for >85% purity) .

How can site-directed mutagenesis be applied to study the catalytic mechanism of SSO0094?

Site-directed mutagenesis is a powerful approach for investigating the catalytic mechanism of enzymes like SSO0094. Based on studies of related phosphatases from Sulfolobus solfataricus, the following methodology can be applied:

Target Residue Selection:

• Catalytic Site Residues: Identify conserved residues likely involved in catalysis through sequence alignment with characterized phosphatases.

• Substrate Binding Residues: Target residues in the predicted substrate binding pocket.

• Potential Regulatory Sites: Investigate serine, threonine, or tyrosine residues that might be subject to phosphorylation-based regulation .

Mutagenesis Strategy:

• Conservative Mutations: Replace residues with similarly sized but catalytically inactive alternatives (e.g., Ser→Ala, Asp→Asn) to test specific chemical roles.

• Charge Mutations: Substitute charged residues to test electrostatic contributions (e.g., Ser→Asp to mimic phosphorylation) .

• Size Variations: Introduce smaller or larger residues to probe spatial constraints (e.g., Ser→Thr) .

Functional Characterization:

• Steady-State Kinetics: Determine kcat and KM parameters for wild-type and mutant enzymes.

• pH-Rate Profiles: Identify ionization states of catalytic residues.

• Substrate Specificity: Test altered specificity resulting from mutations.

As demonstrated with the S. solfataricus phosphohexomutase, replacing Ser-309 with aspartic acid reduced Vmax to only 4% of wild-type activity, while substitution with threonine caused a three- to fivefold decrease . Similar approaches with SSO0094 could reveal key mechanistic details of its catalytic function.

What analytical techniques are most suitable for studying thermal stability of SSO0094?

As a protein from the hyperthermophilic archaeon Sulfolobus solfataricus, which thrives at temperatures around 80°C and pH 2-3, SSO0094 possesses remarkable thermal stability. Several analytical techniques are particularly valuable for characterizing this property:

Differential Scanning Calorimetry (DSC):
DSC directly measures the heat capacity of a protein solution as temperature increases, providing thermodynamic parameters of unfolding:

• Melting Temperature (Tm): The temperature at which 50% of the protein is unfolded.

• Enthalpy of Unfolding (ΔH): The heat absorbed during the unfolding process.

• Heat Capacity Change (ΔCp): Provides information about hydrophobic exposure during unfolding.

Circular Dichroism (CD) Spectroscopy:
CD monitors changes in protein secondary structure with increasing temperature:

• Thermal Melting Curves: Tracking ellipticity at 222 nm (α-helix) or 218 nm (β-sheet) during heating.

• Structural Transitions: Identifying intermediate states during unfolding.

• Reversibility Assessment: Testing refolding capacity after thermal denaturation.

Differential Scanning Fluorimetry (DSF/Thermofluor):
This technique uses environmentally sensitive fluorescent dyes to monitor protein unfolding:

• High-Throughput Screening: Testing multiple buffer conditions simultaneously.

• Ligand Effects: Assessing how substrate binding affects thermal stability.

• pH and Salt Dependencies: Determining optimal conditions for maximum stability.

Activity Assays at Various Temperatures:
Measuring enzyme activity across a temperature range provides functional information:

• Temperature Optimum: Identifying the temperature of maximum activity.

• Activation Energy: Calculating from Arrhenius plots.

• Thermal Inactivation Kinetics: Determining rates of activity loss at different temperatures.

Data from these techniques can be compiled into a comprehensive thermal stability profile, which is crucial for understanding the structural adaptations that allow SSO0094 to function under extreme conditions.