Recombinant Sulfolobus solfataricus 30S ribosomal protein S4P (rps4p)

What taxonomic classification applies to Sulfolobus solfataricus from which rps4p is derived?

Sulfolobus solfataricus is a hyperthermophilic archaeon belonging to the phylum Crenarchaeota within the domain Archaea. The specific strain commonly used in research is ATCC 35092 / DSM 1617 / JCM 11322 / P2 . This organism is characterized by its ability to thrive in extreme environments, particularly at high temperatures (optimal growth at 75-80°C) and acidic conditions (pH 2-3) .

The taxonomic hierarchy is:

• Domain: Archaea

• Phylum: Crenarchaeota

• Class: Thermoprotei

• Order: Sulfolobales

• Family: Sulfolobaceae

• Genus: Sulfolobus

• Species: Sulfolobus solfataricus

Understanding this taxonomic positioning is crucial when designing experiments, as the extremophilic nature of the organism significantly influences the properties of its proteins, including rps4p.

How does archaeal rps4p differ structurally from bacterial and eukaryotic homologs?

Archaeal ribosomal proteins, including S. solfataricus rps4p, represent a unique evolutionary position with distinctive structural features:

In S. solfataricus specifically, rps4p incorporates enhanced hydrophobic packing and increased numbers of salt bridges that contribute to thermostability . These adaptations allow the protein to maintain structural integrity at the high temperatures (75-80°C) required for growth of this hyperthermophilic archaeon.

The RNA-binding interface of archaeal rps4p shows some conservation with bacterial homologs, reflecting the functional requirement for 16S rRNA interaction, but with archaeal-specific modifications that accommodate the unique structure of archaeal ribosomes.

What expression system yields optimal recombinant S. solfataricus rps4p production?

For optimal expression of recombinant S. solfataricus rps4p, a systematic approach addressing multiple parameters is recommended:

Expression System Selection:

Critical Protocol Considerations:

• Media composition: LB medium supplemented with 0.2% glucose helps repress basal expression .

• Temperature management: While S. solfataricus grows optimally at 75-80°C, recombinant expression in E. coli requires lower temperatures (20-30°C) to prevent inclusion body formation.

• Codon optimization: The GC-rich genome of S. solfataricus can create translation inefficiencies in E. coli, making codon optimization or use of Rosetta strains beneficial.

• Cell lysis: Initial heat treatment (70°C for 20 minutes) exploits the thermostability of rps4p to eliminate many E. coli proteins.

Following this optimized protocol typically yields 5-10 mg of purified rps4p per liter of culture with purity exceeding 85% after standard purification steps .

What purification strategy achieves highest purity of functional recombinant rps4p?

A multi-step purification strategy optimized for recombinant S. solfataricus rps4p yields protein with >85% purity suitable for functional and structural studies:

Step 1: Thermal Precipitation

• Heat cell lysate to 70°C for 20 minutes

• Centrifuge at 16,000 × g for 30 minutes to remove denatured E. coli proteins

• This exploits the thermostability of archaeal proteins and serves as an initial purification step

Step 2: Affinity Chromatography

• For His-tagged rps4p, use Ni-NTA chromatography

• Binding buffer: 50 mM phosphate buffer pH 8.0, 300 mM NaCl, 10 mM imidazole

• Elution with 250-300 mM imidazole gradient

• Tag removal if necessary using appropriate protease

Step 3: Ion Exchange Chromatography

• Based on rps4p's high calculated pI (~10), use cation exchange (e.g., SP Sepharose)

• Gradient elution with increasing NaCl concentration (0-1M)

• This step effectively separates nucleic acid contaminants and similarly sized proteins

Step 4: Size Exclusion Chromatography

• Final polishing step using Superdex 75 or equivalent

• Running buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

• Removes aggregates and provides buffer exchange

Quality Control Metrics:

• SDS-PAGE analysis should show a single band at approximately 19 kDa

• Western blot confirmation using anti-His or specific anti-rps4p antibodies

• Typical final purity exceeds 85% as determined by gel densitometry

• Mass spectrometry verification of intact mass and sequence coverage

This protocol can be scaled up for preparative purposes or modified for isotopic labeling for NMR or other biophysical studies.

What storage conditions maintain optimal activity of purified recombinant rps4p?

Proper storage of S. solfataricus rps4p is critical for maintaining structural integrity and functional activity. The following conditions are recommended based on intended use and timeframe:

Short-term Storage (1-2 weeks):

• Temperature: 4°C

• Buffer: 20 mM Tris-HCl pH 7.5, 150 mM NaCl

• Additives: 0.5 mM EDTA and 0.02% sodium azide to prevent microbial contamination

Long-term Storage Options:

• Frozen storage:

  • Temperature: -80°C (preferred) or -20°C

  • Aliquot in small volumes to prevent freeze-thaw cycles

  • Add glycerol to 50% final concentration when storing at -20°C

• Lyophilization:

  • Provides extended shelf life up to 12 months

  • Buffer exchange to volatile buffer (e.g., ammonium bicarbonate) before freeze-drying

  • Store lyophilized powder at -20°C with desiccant

Reconstitution Protocol:

• For lyophilized protein, reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• Addition of 5-50% glycerol after reconstitution improves stability

• Allow complete rehydration (15-30 minutes) before functional assays

Stability Monitoring:

• Verify protein integrity after storage by SDS-PAGE

• For functional verification, use RNA binding assays (EMSA)

• Activity should be assessed before critical experiments, particularly after extended storage

Although S. solfataricus proteins generally exhibit excellent stability due to their thermophilic origin, proper storage conditions remain essential for maintaining full experimental functionality.

What experimental approaches best characterize RNA-binding properties of rps4p?

Comprehensive characterization of rps4p RNA-binding properties requires multiple complementary techniques:

Primary Binding Assessment:

• Electrophoretic Mobility Shift Assay (EMSA)

  • Detects complex formation through mobility changes of labeled RNA

  • Can be performed with radioactively labeled RNA ([32P]) for high sensitivity

  • Non-denaturing PAGE (6-8%) run at room temperature or 4°C

  • Particularly valuable for initial binding confirmation and specificity testing

• Filter Binding Assays

  • Quantitative determination of binding parameters

  • Requires radiolabeled RNA and nitrocellulose membranes

  • Rapid and suitable for multiple condition testing

Quantitative Binding Characterization:

• Surface Plasmon Resonance (SPR)

  • Real-time kinetic analysis (kon and koff rates)

  • No labeling required for one binding partner

  • Temperature control allows binding assessment under near-physiological conditions

• Microscale Thermophoresis (MST)

  • Measures binding in solution with minimal sample consumption

  • Tolerates wide range of buffer conditions

  • Suitable for thermostable proteins like those from S. solfataricus

Structural Analysis of Binding:

• RNA Footprinting

  • Identifies protected RNA regions using chemical or enzymatic probes

  • SHAPE (Selective 2'-Hydroxyl Acylation analyzed by Primer Extension) particularly valuable

  • Can be performed at elevated temperatures to mimic physiological conditions

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS)

  • Maps protein regions involved in RNA binding

  • Provides dynamics information not available from static techniques

For S. solfataricus rps4p specifically, protocols should be adapted to accommodate the thermophilic nature of the protein, potentially including elevated temperature binding conditions to observe physiologically relevant interactions .

How does temperature affect the structural stability and function of recombinant rps4p?

The thermophilic origin of S. solfataricus rps4p results in remarkable temperature-dependent properties that must be considered in experimental design:

Thermal Stability Profile:

Molecular Basis for Thermostability:

• Increased number of salt bridges (ion pairs) compared to mesophilic homologs

• Enhanced hydrophobic core packing

• Reduction in thermolabile residues (Asn, Gln, Met, Cys)

• Higher proportion of charged amino acids on protein surface

Experimental Implications:

• Thermal shift assays (differential scanning fluorimetry) typically show Tm values >85°C for rps4p

• Circular dichroism spectroscopy reveals minimal structural changes between 25-75°C, confirming extraordinary stability

• Activity assays should ideally be performed at 65-75°C to observe native function, though many binding interactions remain detectable at lower temperatures

• Buffer selection requires consideration of temperature effects on pH (especially for Tris buffers)

This exceptional thermal stability makes rps4p an excellent model for studying protein adaptation to extreme environments while also requiring specialized considerations for experimental design compared to mesophilic proteins .

What is the functional role of rps4p in archaeal ribosome assembly and translation?

S. solfataricus rps4p serves multiple critical functions in archaeal ribosome biogenesis and protein synthesis:

Ribosomal Assembly Functions:

• Acts as a primary binding protein that directly interacts with 16S rRNA

• Forms a nucleation center for subsequent 30S subunit assembly

• Stabilizes critical rRNA tertiary structures

• May coordinate assembly with cellular metabolism through its dual binding properties

Translational Functions:

• Contributes to mRNA binding and positioning within the decoding center

• Enhances translational fidelity

• Provides structural stability to the assembled 30S subunit, especially critical at high temperatures

• Participates in ribosome-associated quality control mechanisms

Archaeal-Specific Roles:

• In thermophilic species like S. solfataricus, rps4p contains additional stabilizing elements that maintain ribosome integrity at elevated temperatures

• May interact with archaeal-specific translation factors

• Contains binding domains for specialized archaeal RNAs, potentially including C/D box small RNAs

This multifunctional role positions rps4p as both a structural component and functional participant in archaeal translation, making it an important model for understanding the evolution of translation machinery across domains of life and adaptation to extreme environments .

How can structural biology techniques characterize rps4p integration into the archaeal 30S subunit?

Multiple structural biology approaches provide complementary insights into rps4p's incorporation into archaeal 30S ribosomal subunits:

Cryo-Electron Microscopy (Cryo-EM):

• Achieves near-atomic resolution of intact ribosomal complexes

• Visualization of rps4p within the native 30S context

• Sample preparation should maintain physiological conditions for thermophilic archaea

• Time-resolved cryo-EM can capture assembly intermediates

• Expected resolution: 2.5-3.5Å for well-ordered regions

X-ray Crystallography:

• High-resolution structure of isolated rps4p (potential resolution <2.0Å)

• Co-crystallization with specific rRNA fragments identifies precise binding interfaces

• Crystallization conditions must account for thermostability (higher salt, temperature considerations)

Nuclear Magnetic Resonance (NMR):

• Dynamic information about rps4p flexibility and binding-induced conformational changes

• TROSY-based methods overcome size limitations for specific domains

• Chemical shift perturbation experiments map RNA binding interfaces

Integrative Structural Biology Workflow:

• Initial characterization: Obtain high-resolution structure of isolated rps4p

• Binding interface mapping: Identify rRNA interaction sites through footprinting and crosslinking

• Complex visualization: Determine position in assembled 30S subunit via cryo-EM

• Dynamics assessment: Characterize conformational changes upon binding

• Validation: Confirm key interactions through mutagenesis and functional assays

This multi-technique approach provides comprehensive structural insights into how rps4p contributes to archaeal ribosome architecture and function, particularly in the context of adaptation to extreme environments like those inhabited by S. solfataricus .

What methodologies can identify protein-protein interactions between rps4p and other ribosomal components?

Identifying protein-protein interactions involving rps4p requires specialized approaches that accommodate the thermophilic nature of S. solfataricus proteins:

In Vitro Interaction Methods:

In Vivo Approaches:

• Proximity-dependent biotin identification (BioID)

  • Modified for expression in Sulfolobus species

  • Temperature-stable biotin ligase variants required

  • Identifies physiologically relevant interactions

• Co-immunoprecipitation (Co-IP)

  • Using antibodies against rps4p or epitope-tagged versions

  • Must maintain native buffer conditions mimicking cytoplasmic environment

  • Western blot or mass spectrometry identification of binding partners

Reconstitution Studies:

• In vitro assembly of 30S subunits with labeled components

• Systematic omission experiments to establish dependency relationships

• FRET-based approaches to measure distances between specific proteins

Bioinformatic Prediction and Validation:

• Coevolution analysis to predict interacting partners

• Molecular docking guided by experimental constraints

• Validation through targeted mutagenesis of predicted interfaces

These complementary approaches provide a comprehensive characterization of the protein-protein interaction network involving rps4p in the archaeal 30S subunit, accounting for the unique challenges presented by thermophilic proteins .

How do post-translational modifications affect S. solfataricus rps4p function?

Post-translational modifications (PTMs) of S. solfataricus rps4p represent an emerging research area with significant implications for archaeal ribosome regulation:

Known and Predicted PTMs in Archaeal Ribosomal Proteins:

Analytical Approaches:

• Mass Spectrometry-Based PTM Profiling:

  • Bottom-up proteomics with enrichment strategies for specific modifications

  • Top-down proteomics for intact protein analysis

  • Quantitative approaches to compare modification levels under different conditions

• Functional Impact Assessment:

  • Site-directed mutagenesis of modification sites

  • In vitro translation assays comparing modified and unmodified forms

  • Ribosome assembly studies with modification-mimicking variants

• Structural Consequences:

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational effects

  • Molecular dynamics simulations comparing modified and unmodified states

  • Binding assays to quantify effects on RNA and protein interactions

The extreme growth conditions of S. solfataricus may necessitate unique PTM patterns not observed in mesophilic organisms, potentially contributing to ribosomal adaptation to high temperature and acidic environments .

What evolutionary insights emerge from comparative analysis of archaeal rps4p sequences?

Evolutionary analysis of rps4p across the archaeal domain reveals important insights into ribosomal protein evolution and adaptation to extreme environments:

Phylogenetic Distribution and Conservation:

Thermal Adaptation Signatures:

Analysis of amino acid composition across thermophilic, mesophilic, and psychrophilic archaeal rps4p sequences reveals:

• Increased Glu+Lys/Gln+His ratio correlates with optimal growth temperature

• Higher proportion of charged residues in thermophiles

• Reduction in thermolabile residues (Asn, Gln, Met) in hyperthermophiles

• Distinctive patterns of predicted hydrogen bonding networks

Coevolution with rRNA:

• Compensatory mutations in protein and rRNA maintain critical binding interfaces

• Co-variation analysis identifies nucleotide-amino acid pairs under selection pressure

• RNA-binding domains show greater conservation than other regions

These evolutionary patterns provide crucial context for understanding S. solfataricus rps4p's structure-function relationships and offer insights into the adaptation of translational machinery to extreme environments across the archaeal domain .

How can computational approaches predict interactions between rps4p and archaeal translation factors?

Advanced computational methods offer valuable tools for predicting interactions between S. solfataricus rps4p and archaeal translation factors:

Computational Prediction Pipeline:

• Structure Preparation:

  • Homology modeling of proteins lacking experimental structures

  • Integration of available cryo-EM/crystal structure data for rps4p

  • Model refinement using molecular dynamics simulations under conditions mimicking thermophilic environments

• Interface Prediction:

  • Machine learning algorithms trained on known archaeal protein interfaces

  • Conservation mapping to identify functionally important surfaces

  • Electrostatic complementarity analysis (particularly important for thermophilic proteins)

• Protein-Protein Docking:

  • Rigid body docking followed by flexible refinement

  • Scoring functions optimized for archaeal protein interactions

  • Ensemble approaches to account for conformational flexibility

• Molecular Dynamics Validation:

  • Simulations at elevated temperatures (70-80°C) to mimic physiological conditions

  • Stability assessment of predicted complexes

  • Free energy calculations to estimate binding affinities

Example Prediction Workflow for rps4p-aIF2 Interaction:

Experimental Validation Strategies:

• Site-directed mutagenesis of predicted interface residues

• In vitro binding assays under native temperature conditions

• Crosslinking mass spectrometry targeting predicted interfaces

This computational framework generates testable hypotheses about rps4p interactions with archaeal translation machinery, particularly valuable for understanding the unique features of hyperthermophilic translation systems .

How should experiments be designed to account for the thermophilic nature of S. solfataricus rps4p?

The hyperthermophilic origin of S. solfataricus rps4p necessitates specialized experimental designs:

Temperature Considerations:

• Optimal functional assays should be conducted at 65-75°C to reflect native conditions

• Temperature-controlled equipment is essential (thermocyclers, incubators, spectrophotometers)

• Some assays may require temperature compromises (e.g., 50-60°C) to balance equipment limitations with protein activity

Buffer Stability:

• Use temperature-stable buffers (phosphate, HEPES) rather than Tris, which has high temperature coefficients

• Increase buffer concentration (50-100 mM) to account for pH shifts at elevated temperatures

• Include stabilizing agents (glycerol, trehalose) for prolonged high-temperature incubations

Control Design:

• Include mesophilic homologs as comparative controls

• Implement temperature gradients to establish activity profiles

• Include thermal stability controls in all functional assays

Specialized Equipment Needs:

• High-temperature spectrophotometers for real-time assays

• Thermostable microplates for high-throughput experiments

• Temperature-controlled chambers for binding studies

By accounting for these thermophilic adaptations in experimental design, researchers can generate more physiologically relevant data and better understand the unique properties of S. solfataricus rps4p in its native context.