Recombinant Sulfolobus islandicus rod-shaped virus 1 Uncharacterized protein 268 (268)

What is Sulfolobus islandicus rod-shaped virus 1 and why is Uncharacterized protein 268 significant for research?

Sulfolobus islandicus rod-shaped virus 1 (SIRV-1) is an archaeal virus that infects hyperthermophilic acidophiles of the genus Sulfolobus, which thrive in extreme environments with temperatures of 75-85°C and pH levels of 2-3. Uncharacterized protein 268 (268 amino acids in length) represents one of the viral proteins whose function remains to be fully elucidated. This protein is significant for research because it provides insights into viral adaptation to extreme environments and potential novel biochemical properties associated with thermostability .

The significance of studying this protein extends beyond virology, as proteins from extremophilic organisms often possess unique structural features and enzymatic properties that may have biotechnological applications. Understanding the structure-function relationship of this protein could reveal novel biological mechanisms employed by viruses to survive in extreme conditions.

What are the optimal storage conditions for maintaining the activity of Recombinant Sulfolobus islandicus rod-shaped virus 1 Uncharacterized protein 268?

For optimal storage of this recombinant protein, the following conditions should be maintained:

Repeated freeze-thaw cycles significantly compromise protein integrity and should be avoided. Instead, prepare small aliquots during initial handling to minimize the need for multiple freeze-thaw events . The high glycerol content (50%) in the storage buffer is critical for maintaining protein stability by preventing ice crystal formation that could denature the protein structure.

How should researchers validate the identity and purity of the recombinant protein before experimental use?

Validation of identity and purity should follow a multi-method approach:

• SDS-PAGE analysis to confirm the expected molecular weight (~30 kDa based on the 268 amino acid sequence)

• Western blotting using antibodies against the protein or any fusion tags

• Mass spectrometry for precise molecular weight confirmation and peptide mapping

• Circular dichroism to assess secondary structure integrity

• Size exclusion chromatography to evaluate oligomeric state and homogeneity

For quantitative experiments, protein concentration should be determined using multiple methods, including:

• Bradford or BCA assay for total protein concentration

• UV absorbance at 280 nm (using the calculated extinction coefficient based on aromatic amino acid content)

• Amino acid analysis for absolute quantification

These validation steps are essential prerequisites for ensuring experimental reproducibility and reliable data interpretation.

What experimental approaches are recommended for determining the potential function of this uncharacterized viral protein?

A comprehensive functional characterization strategy should incorporate several complementary approaches:

Structural Analysis Methods:

• X-ray crystallography or Cryo-EM to determine three-dimensional structure

• NMR spectroscopy for dynamic aspects and interaction sites

• Small-angle X-ray scattering (SAXS) for solution structure information

Functional Screening Approaches:

• Protein-protein interaction studies using pull-down assays, yeast two-hybrid, or proximity labeling

• DNA/RNA binding assays (EMSA, filter binding assays) to test nucleic acid interaction capacity

• Enzymatic activity screening against various substrates

• Host proteome interaction studies using co-immunoprecipitation followed by mass spectrometry

In silico Analysis:

• Structural homology modeling and comparison with known protein structures

• Sequence-based predictive algorithms for potential functional domains

• Molecular dynamics simulations to predict stability at high temperatures

The integration of these methodologies provides a robust framework for functional hypothesis generation and testing. When implemented sequentially, starting with computational predictions followed by biochemical validation, this approach maximizes resource efficiency while systematically narrowing potential functional assignments .

How can researchers effectively develop assays to study the thermostability properties of this archaeal viral protein?

Developing effective thermostability assays for this archaeal viral protein requires consideration of its extreme environment origin. The following methodological framework is recommended:

Differential Scanning Calorimetry (DSC) Protocol:

• Prepare protein samples at 0.5-1.0 mg/mL in appropriate buffers

• Perform temperature scans from 25°C to 95°C at a rate of 1°C/minute

• Calculate melting temperature (Tm) and enthalpy of unfolding

• Compare Tm values across different buffer conditions to optimize stability

Circular Dichroism (CD) Thermal Denaturation:

• Record CD spectra at increasing temperatures (25°C to 95°C)

• Monitor changes in secondary structure elements

• Plot fractional denaturation versus temperature to determine transition points

Activity Retention Assays:

• Incubate protein aliquots at various temperatures (60°C, 70°C, 80°C, 90°C)

• Remove samples at defined time intervals (5, 15, 30, 60, 120 minutes)

• Measure residual activity or structural integrity

• Calculate half-life at each temperature

Comparative Analysis Framework:

• Include mesophilic protein controls in parallel experiments

• Normalize data to initial activity/structure measurements

• Generate Arrhenius plots to calculate activation energy of denaturation

These approaches provide quantitative metrics for protein thermostability and establish a foundation for structure-function studies relating to environmental adaptation mechanisms.

What is the recommended methodology for expressing and purifying Recombinant Sulfolobus islandicus rod-shaped virus 1 Uncharacterized protein 268?

The expression and purification of this archaeal viral protein presents unique challenges due to its extremophilic origin. A recommended protocol includes:

Expression System Selection:

• E. coli BL21(DE3) with codon optimization for archaeal codon usage

• Alternative consideration: Thermophilic expression hosts for proper folding

• Vector selection incorporating heat-stable selection markers

Optimized Expression Protocol:

• Transform expression construct into host cells

• Culture cells at 37°C to OD600 of 0.6-0.8

• Induce with 0.5 mM IPTG

• Shift temperature to 30°C for 4-6 hours (to balance yield with solubility)

• Harvest cells by centrifugation (5,000 × g, 15 minutes, 4°C)

Purification Strategy:

• Resuspend cells in lysis buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole)

• Lyse cells using sonication or pressure-based methods

• Heat treatment (65°C for 20 minutes) to leverage natural thermostability

• Remove precipitated host proteins by centrifugation (20,000 × g, 30 minutes, 4°C)

• Perform immobilized metal affinity chromatography (IMAC)

• Apply size exclusion chromatography for final polishing

• Concentrate protein and exchange into storage buffer

Yield Assessment:

• Typical yield: 5-15 mg purified protein per liter of bacterial culture

• Purity confirmation by SDS-PAGE (>95% homogeneity)

• Functional validation through activity assays or structural analysis

This methodology emphasizes utilizing the intrinsic thermostability of the protein as a purification advantage, allowing for significant removal of contaminating E. coli proteins through heat precipitation steps.

How can researchers investigate potential protein-protein interactions between Uncharacterized protein 268 and host cellular components?

Investigating protein-protein interactions between this viral protein and host components requires specialized approaches accounting for the extremophilic nature of both virus and host. A comprehensive investigation should include:

In vitro Interaction Analysis:

• Recombinant expression of Sulfolobus cellular proteins

• Pull-down assays using immobilized Uncharacterized protein 268

• Surface plasmon resonance (SPR) for binding kinetics determination

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Microscale thermophoresis for interaction studies under varied conditions

In vivo Approaches:

• Proximity-dependent biotin identification (BioID) with protein 268 as bait

• Co-immunoprecipitation coupled with mass spectrometry (IP-MS)

• Fluorescence resonance energy transfer (FRET) with fluorescently labeled proteins

• Split-reporter protein complementation assays

Comparative Interaction Analysis:

• Test interactions at different temperatures (37°C, 60°C, 80°C)

• Evaluate pH-dependent interaction profiles (pH 2-7)

• Compare interaction networks with related archaeal viruses

The identified interactions should be validated through multiple independent methods and integrated with structural data to map interaction interfaces. This multi-faceted approach addresses the challenge of studying extremophilic protein interactions under physiologically relevant conditions while minimizing artifacts.

What techniques can be used to study the structural characteristics of Uncharacterized protein 268 under extremophilic conditions?

Studying protein structure under extremophilic conditions requires specialized approaches:

High-Temperature Structural Analysis:

• Temperature-controlled X-ray crystallography (20-80°C)

• NMR spectroscopy with variable temperature experiments

• High-temperature CD spectroscopy with specialized cells

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) at elevated temperatures

Acidic pH Structural Studies:

• Crystallization screens at pH 2-4

• Solution NMR at acidic pH with internal standards

• Acidic pH-resistant sample cells for biophysical measurements

Comparative Structural Data Collection:

Structure Stabilization Strategies:

• Chemical crosslinking to capture transient states

• Nanodiscs or amphipols for membrane-interacting regions

• Deuterium oxide (D₂O) buffer to minimize radiation damage

These approaches collectively enable structural characterization under conditions mimicking the natural archaeal host environment, providing insights into protein adaptations to extreme conditions .

How should researchers design comparative studies between Uncharacterized protein 268 and homologous proteins from related viruses or hosts?

Designing effective comparative studies requires systematic sequence and structural analyses followed by functional comparisons:

Sequence-Based Comparative Analysis:

• Perform PSI-BLAST searches against archaeal viral databases

• Use HHpred and other hidden Markov model approaches to detect distant homologs

• Construct multiple sequence alignments with MAFFT or MUSCLE

• Generate phylogenetic trees to understand evolutionary relationships

• Identify conserved motifs and variable regions

Structural Comparison Methodology:

• Generate structural models using AlphaFold or RoseTTAFold

• Perform structural alignments using DALI or TM-align

• Quantify structural conservation using RMSD and GDT scores

• Map conservation onto structures to identify functional hotspots

Functional Conservation Assessment:

• Express and purify homologous proteins using identical protocols

• Compare thermostability profiles under standardized conditions

• Assess binding properties to identical substrates/partners

• Measure enzymatic parameters if applicable

Data Integration Framework:

What strategies can address solubility issues when working with Recombinant Sulfolobus islandicus rod-shaped virus 1 Uncharacterized protein 268?

Solubility challenges with this archaeal viral protein often stem from its adaptation to extreme environments. Systematic troubleshooting approaches include:

Expression Optimization Strategies:

• Lower induction temperature (16-25°C)

• Reduce inducer concentration (0.1-0.3 mM IPTG)

• Use auto-induction media for gradual protein expression

• Co-express with archaeal chaperones (e.g., thermosome components)

• Test multiple fusion tags (MBP, SUMO, TrxA) known to enhance solubility

Buffer Optimization Protocol:

• Screen buffers across pH range 4.0-8.0

• Test different salt concentrations (100-500 mM NaCl)

• Evaluate stabilizing additives:

Refolding Strategies:

• Solubilize inclusion bodies in 8M urea or 6M guanidine HCl

• Perform stepwise dialysis at elevated temperatures (40-60°C)

• Apply on-column refolding during affinity purification

• Use artificial chaperone-assisted refolding with cyclodextrin

Structural Modification Approaches:

• Truncate flexible termini based on disorder predictions

• Design solubility-enhancing point mutations at surface residues

• Create chimeric constructs with soluble archaeal proteins

Implementation of these strategies should follow a decision tree approach, starting with expression optimization before proceeding to more labor-intensive refolding procedures.

How can researchers address protein degradation issues during purification of the Uncharacterized protein 268?

Protein degradation during purification requires a systematic troubleshooting approach:

Preventative Measures During Lysis:

• Maintain samples at 4°C throughout processing

• Include protease inhibitor cocktail optimized for archaeal proteases

• Add EDTA (1-5 mM) to inhibit metalloproteases

• Perform all steps rapidly to minimize exposure time

Degradation Analysis and Identification:

• N-terminal sequencing of degradation fragments

• Mass spectrometry to identify cleavage sites

• In-gel digestion and peptide mapping

• Western blotting with antibodies against different regions

Purification Strategy Modifications:

• Incorporate ion exchange chromatography steps to separate degradation products

• Use affinity tags at both N- and C-termini to ensure full-length purification

• Apply hydrophobic interaction chromatography to separate partially degraded species

• Perform preparative SEC as final polishing step

Stability Enhancement:

• Identify and modify protease-susceptible sites through mutagenesis

• Optimize buffer conditions based on thermal shift assays

• Include stabilizing additives (glycerol, arginine, proline)

• Store purified protein with reversible protease inhibitors

The effectiveness of these interventions should be quantitatively assessed by monitoring degradation rates using SDS-PAGE and densitometry analysis under various conditions. Systematic implementation of these strategies typically reduces degradation by 80-90% in challenging archaeal proteins.

What approaches can resolve aggregation issues when working with concentrated solutions of this recombinant protein?

Protein aggregation is a common challenge when working with concentrated solutions of recombinant proteins. For Uncharacterized protein 268, the following methodological approaches can mitigate aggregation:

Aggregation Detection and Characterization:

• Dynamic light scattering (DLS) to monitor particle size distribution

• Analytical ultracentrifugation (AUC) to quantify aggregate population

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS)

• Thioflavin T binding assays to detect amyloid-like aggregates

Buffer Optimization Strategy:

• pH screening (0.5 unit increments) around the protein's theoretical pI

• Ionic strength titration (50-500 mM NaCl)

• Addition of solubilizing agents:

Concentration Methodology Modifications:

• Implement stepwise concentration with monitoring at each step

• Use dialysis-based concentration against high molecular weight PEG

• Perform concentration at elevated temperatures (30-40°C)

• Add 10% glycerol during concentration process

Structural Engineering Approaches:

• Identify and mutate surface-exposed hydrophobic residues

• Introduce charged residues at aggregation-prone interfaces

• Design stabilizing disulfide bonds based on structural models

• Create fusion constructs with highly soluble partners

By implementing these approaches systematically and monitoring aggregation using multiple orthogonal techniques, researchers can typically achieve protein concentrations of 5-10 mg/mL without significant aggregation, enabling structural and functional studies that require concentrated protein solutions .

What mass spectrometry approaches are most suitable for characterizing post-translational modifications of Uncharacterized protein 268?

Characterizing post-translational modifications (PTMs) of archaeal viral proteins requires specialized mass spectrometry approaches:

Sample Preparation Strategies:

• Multiple proteolytic digestions (trypsin, chymotrypsin, Glu-C) for comprehensive coverage

• Enrichment techniques for specific modifications:

  • TiO₂ chromatography for phosphopeptides

  • Hydrazide chemistry for glycopeptides

  • Antibody-based enrichment for acetylation, methylation

• Chemical derivatization to enhance ionization efficiency of modified peptides

MS Instrumentation and Methods:

Data Analysis Workflow:

• Database searches allowing for variable modifications

• De novo sequencing for unexpected modifications

• Neutral loss scanning for phosphorylation (−98 Da)

• Extracted ion chromatograms for modification site localization

• Statistical validation using false discovery rate calculation

Comparative Analysis Framework:

• Compare PTM profiles across different expression systems

• Analyze modification changes under different temperature/pH conditions

• Correlate modifications with functional properties

• Map modification sites onto structural models

These comprehensive MS approaches can identify archaeal-specific modifications such as methylation, acetylation, and unique glycosylation patterns that may contribute to protein thermostability and function in extreme environments.

How can researchers effectively use computational approaches to predict the function of Uncharacterized protein 268?

Computational prediction of protein function should follow a multi-layered approach integrating various predictive methods:

Sequence-Based Prediction Pipeline:

• Conserved domain identification using CDD, SMART, and Pfam

• Motif detection using PROSITE and ELM

• Remote homology detection using HHpred and HMMER

• Functional site prediction (active sites, binding pockets)

• Disordered region prediction and functional analysis

Structure-Based Prediction Methods:

• Generate 3D models using AlphaFold2 or RoseTTAFold

• Structural alignment against PDB using DALI and TM-align

• Binding site prediction with CASTp and POCKET

• Molecular docking with potential substrates/partners

• Molecular dynamics simulations at high temperatures

Integrated Function Prediction:

• Gene neighborhood analysis in archaeal viral genomes

• Protein-protein interaction network prediction

• Gene Ontology term assignment using multiple tools

• Enzyme classification using machine learning approaches

Validation and Refinement Framework:

The confidence of functional predictions can be quantitatively assessed using scoring schemes that incorporate:

• Conservation scores across related viruses

• Structural similarity Z-scores

• Template modeling scores for structural models

• Consensus across multiple prediction methods

This integrated computational approach typically achieves 60-80% accuracy in broad functional assignment and can generate testable hypotheses for experimental validation .

What are the appropriate controls and standards for biophysical characterization experiments with this archaeal viral protein?

Rigorous biophysical characterization requires appropriate controls and standards:

Essential Controls for Biophysical Experiments:

• Negative Controls:

  • Buffer-only measurements to establish baselines

  • Heat-denatured protein samples as unfolded references

  • Non-binding protein variants (if available)

  • Unrelated proteins of similar size/structure

• Positive Controls:

  • Well-characterized archaeal proteins with known properties

  • Commercial standards with certified values

  • Previously characterized domains from related viruses

  • Engineered constructs with predictable properties

Standard Protocols for Key Biophysical Techniques:

Quantitative Data Analysis Requirements:

• Replicate measurements (minimum n=3)

• Statistical analysis of fitted parameters

• Error propagation through derived values

• Comparison with literature values for similar proteins

Quality Control Metrics:

• Sample monodispersity verification before experiments

• Concentration determination by multiple methods

• Post-experiment sample integrity confirmation

• Instrument performance validation with standards

Implementing these controls and standards ensures data reliability and facilitates meaningful comparison with other studies. For archaeal proteins, special consideration should be given to temperature effects on both the protein and the measurement system itself.

What are the potential biotechnological applications of Uncharacterized protein 268 based on its extremophilic origin?

The extremophilic origin of this archaeal viral protein suggests several promising biotechnological applications:

Enzyme Technology Applications:

• Thermostable biocatalysts for high-temperature industrial processes

• Acid-resistant enzymatic activities for food processing or biofuel production

• Novel activities for specialized chemical transformations

• Detergent additives requiring stability in harsh conditions

Biomaterial Development:

• Protein-based scaffolds for high-temperature applications

• Self-assembling nanostructures from thermostable domains

• Biosensors functional in extreme environments

• Thermostable protein coatings for medical devices

Research Tool Development:

Pharmaceutical Applications:

• Drug delivery systems with enhanced stability

• Thermostable vaccine components

• Novel antimicrobial strategies targeting archaeal systems

The development pathway for these applications requires:

• Full structural and functional characterization

• Protein engineering to optimize desired properties

• Scale-up production optimization

• Application-specific performance testing

Additionally, understanding the fundamental biochemical properties that confer extreme stability could inform the design of synthetic proteins with enhanced resilience to challenging environmental conditions .

How can researchers design experiments to elucidate the role of Uncharacterized protein 268 in viral infection of Sulfolobus hosts?

Designing experiments to elucidate the protein's role in viral infection requires a multi-faceted approach:

Host-Virus Interaction Studies:

• Develop fluorescently labeled protein for localization during infection

• Generate antibodies against the protein for immunolocalization

• Create gene deletion mutants (if genetic system available)

• Perform time-course proteomics during infection cycle

Infection Inhibition Assays:

• Pre-incubate host cells with recombinant protein

• Test neutralizing antibodies against the protein

• Design competitive inhibitors based on structural insights

• Perform complementation studies with mutant variants

Host Receptor Identification Protocol:

• Protein crosslinking with host surface components

• Affinity purification using immobilized protein as bait

• Surface plasmon resonance with host membrane fractions

• Yeast surface display for receptor identification

Functional Assessment Framework:

These approaches collectively address the challenge of studying viral proteins in archaeal systems where genetic tools may be limited. The integration of structural, biochemical, and cell biological approaches provides complementary evidence for functional roles in the infection process.

What interdisciplinary approaches can advance our understanding of archaeal virus-host interactions using Uncharacterized protein 268 as a model?

Advancing understanding of archaeal virus-host interactions requires integrating multiple disciplines:

Integrated Omics Approaches:

• Transcriptomics of host response to purified protein

• Proteomics to identify interacting host partners

• Metabolomics to detect metabolic changes upon protein expression

• Systems biology modeling of virus-host interaction networks

Structural Biology Integration:

• Cryo-electron tomography of infected cells

• In-cell NMR to study protein behavior in native context

• Single-particle tracking in live archaeal cells

• Integrative structural modeling incorporating diverse data types

Evolutionary Bioinformatics:

• Phylogenetic analysis across archaeal virus families

• Evolutionary rate analysis of viral proteins

• Host-range correlation with protein sequence variants

• Ancestral sequence reconstruction and functional testing

Cross-Domain Collaboration Framework:

This interdisciplinary approach addresses the major challenge in archaeal virology—the limited availability of genetic and molecular tools—by leveraging complementary methodologies from multiple fields. The resulting integrated understanding can reveal fundamental principles of virus-host interactions in extremophilic environments that may have broader implications for understanding viral adaptation mechanisms .