Recombinant Desulfotalea psychrophila 50S ribosomal protein L18 (rplR)

What is Desulfotalea psychrophila and why is its ribosomal machinery of interest?

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that thrives at temperatures below 0°C and inhabits permanently cold marine sediments. First discovered in arctic marine sediment off the coast of Svalbard, this gram-negative bacterium plays a significant role in global carbon and sulfur cycles .
D. psychrophila's genome was the first sequenced psychrophilic bacterium (2004), consisting of a 3,523,383 bp circular chromosome with 3,118 predicted genes and two plasmids of 121,586 bp and 14,663 bp . Its ribosomal machinery is of particular interest because it functions efficiently at low temperatures, suggesting specialized adaptations that enable translation in cold environments—making its components valuable models for understanding cold-adapted protein biosynthesis systems.

What functional role does the 50S ribosomal protein L18 play in ribosomes?

The L18 protein is a critical component of the 50S ribosomal subunit and performs essential functions in ribosome assembly and activity:

• It is required for incorporation of 5S rRNA into the ribosome

• It interacts with both 23S rRNA and 5S rRNA, forming part of the central protuberance of the ribosome

• Together with ribosomal protein L5, it can associate into a quaternary complex with 5S and 23S rRNAs

• It serves as one of the primary rRNA binding proteins in the ribosomal assembly

• It likely mediates the attachment of the 5S RNA into the large ribosomal subunit
  This protein is encoded by the rplR gene and belongs to the L18/L5e family of ribosomal proteins, which are highly conserved across bacterial species.

What expression systems are most effective for producing recombinant D. psychrophila ribosomal proteins?

Based on studies with similar psychrophilic proteins, low-temperature expression systems show particular promise for D. psychrophila proteins. A notable approach involves using the cold-adapted bacterium Shewanella sp. strain Ac10 as a host organism with the LI3 promoter .
This system has successfully expressed several D. psychrophila proteins, achieving yields between 5.4-48 mg/liter of culture at 18°C and 1.7-25 mg/liter at 4°C for various proteins . The efficacy of this expression system is demonstrated in the following yield data:

What challenges are specific to expressing psychrophilic ribosomal proteins like D. psychrophila rplR?

Expressing psychrophilic ribosomal proteins presents several unique challenges:

• Thermolability: Proteins from D. psychrophila are generally thermolabile , requiring lower expression temperatures to maintain proper folding and functionality.

• Integration with host ribosomes: Ribosomal proteins may integrate into the host's ribosomes, potentially disrupting translation and resulting in toxicity or growth inhibition.

• Solubility concerns: At higher temperatures, psychrophilic proteins often aggregate or misfold, requiring careful temperature control throughout expression and purification.

• Structural integrity: The proper folding of ribosomal proteins frequently depends on interactions with rRNA and other ribosomal proteins, making isolated expression challenging.

• Codon usage: Differences in codon bias between D. psychrophila and expression hosts may require codon optimization for efficient translation.
  To address these challenges, researchers should consider:

• Using cold-adapted bacterial hosts like Shewanella sp. strain Ac10

• Employing tightly regulated promoters to control expression levels

• Co-expressing with relevant binding partners (e.g., L5 protein) to improve stability

• Including appropriate molecular chaperones in the expression system

How does D. psychrophila rplR interact with other components of the ribosomal complex?

Analysis of protein interaction networks reveals D. psychrophila rplR (DP2776) has significant interactions with:

• Other ribosomal proteins: Strong interaction coefficients (>0.64) with:

  • rplC (50S protein L3, score: 0.647)

  • rplN (50S protein L14, score: 0.651)

  • rpmC (50S protein L29, score: 0.651)

  • rplM (50S protein L13, score: 0.647)

  • rplB (50S protein L2, score: 0.642)

• RNA molecules: Like other L18 proteins, D. psychrophila rplR likely binds to:

  • 5S rRNA (primary interaction)

  • 23S rRNA (as part of the quaternary complex)

• Signal recognition pathways: Functional connections with components of the protein translocation machinery:

  • ffh (signal recognition particle protein, score: 0.857)

  • ftsY (cell division protein FtsY, score: 0.952)

• Motility-related proteins: Unexpected interactions with gliding motility proteins:

  • DP2777 (gliding motility regulatory protein MglB, score: 0.998)

  • DP2775 (unknown protein, potentially motility-related, score: 0.812)
    These interactions suggest D. psychrophila rplR functions both in the ribosomal complex and potentially in ribosome-associated signaling pathways specific to cold-adapted bacteria.

What structural features might contribute to the cold adaptation of D. psychrophila rplR?

While specific structural data for D. psychrophila rplR is not detailed in the search results, typical cold-adapted proteins demonstrate several characteristic structural modifications:

• Increased flexibility: Higher proportion of glycine residues and fewer proline and arginine residues compared to mesophilic homologs

• Reduced hydrophobic core packing: Fewer large hydrophobic amino acids to increase internal flexibility

• Decreased ion pairs and hydrogen bonds: Reduced structural stabilization to maintain flexibility at low temperatures

• Surface charge modifications: Altered surface charge distribution to maintain solubility in cold environments

• Active site adaptations: Modifications that reduce activation energy barriers for catalysis at low temperatures
  Examining these features in D. psychrophila rplR would require comparative structural analysis with mesophilic homologs using techniques such as X-ray crystallography, NMR spectroscopy, or computational structural prediction.

How can recombinant D. psychrophila rplR be utilized to study cold-adapted translation mechanisms?

Recombinant D. psychrophila rplR offers several avenues for investigating cold-adapted translation:

• In vitro reconstitution studies: Comparing ribosome assembly with D. psychrophila rplR versus mesophilic L18 proteins at various temperatures can reveal adaptation mechanisms. Researchers should:

  • Prepare hybrid ribosomes containing D. psychrophila rplR with other components from mesophilic bacteria

  • Assess assembly efficiency and stability at temperature ranges from 0-37°C

  • Measure translation rates of test mRNAs using these hybrid ribosomes

• Binding kinetics experiments: Quantifying the thermodynamics of rplR interactions with rRNA at different temperatures:

  • Use surface plasmon resonance or isothermal titration calorimetry

  • Compare binding constants (Kd), association/dissociation rates (kon/koff), and free energy changes (ΔG)

  • Examine entropy-enthalpy compensation mechanisms potentially unique to psychrophilic proteins

• Complementation assays: Testing whether D. psychrophila rplR can functionally substitute for the L18 protein in mesophilic bacteria at lower temperatures:

  • Create conditional knockout strains of E. coli rplR

  • Complement with D. psychrophila rplR under temperature stress

  • Measure growth rates and translation fidelity at various temperatures

• Stress response studies: Similar to the approach used for PgRL18/L5e , examine expression patterns of D. psychrophila rplR under various stress conditions to understand regulatory mechanisms specific to psychrophilic translation.

What potential biotechnological applications exist for recombinant D. psychrophila rplR?

While avoiding commercial applications as requested, several biotechnological research applications can be considered:

• Cold-active in vitro translation systems: Developing cell-free protein synthesis systems functioning at low temperatures (4-15°C):

  • Incorporate D. psychrophila ribosomal components including rplR

  • Optimize buffer conditions for low-temperature activity

  • Test expression of thermolabile proteins that aggregate at higher temperatures

• Cryopreservation enhancement: Investigating whether D. psychrophila ribosomal proteins confer cryoprotective properties:

  • Test the addition of recombinant rplR to freezing buffers for biological samples

  • Assess protein stabilization effects during freeze-thaw cycles

  • Measure the prevention of aggregation for sensitive enzymes

• Structural biology tools: Using cold-adapted ribosomal proteins as crystallization chaperones:

  • Co-crystallize difficult-to-crystallize proteins with D. psychrophila rplR

  • Explore potential improvements in crystal quality at lower temperatures

  • Develop novel fusion constructs with rplR for enhanced protein solubility

• Educational research kits: Developing comparative biochemistry teaching tools utilizing recombinant D. psychrophila rplR and mesophilic counterparts to demonstrate temperature adaptation principles.

What are optimal conditions for studying D. psychrophila rplR stability and activity?

Based on D. psychrophila's psychrophilic nature and information from similar proteins, the following experimental conditions are recommended:
Temperature conditions:

• Stability studies: 0-25°C range with 5°C increments

• Activity assays: Primary testing at 4-12°C with controls at 25°C and 37°C

• Denaturation studies: Start from 0°C with slow incremental increases
  Buffer recommendations:

• Base buffer: 20-50 mM Tris-HCl or phosphate buffer, pH 7.0-7.5

• Salt concentration: 50-200 mM NaCl or KCl (test multiple concentrations)

• Stabilizing agents: 5-10% glycerol, 1-5 mM MgCl₂, 0.1-1 mM DTT

• Avoid: Detergents at concentrations higher than CMC, chelating agents that may disrupt metal-ion interactions
  Storage guidelines:

• Short-term: 4°C in buffer with 20% glycerol

• Long-term: -80°C with cryoprotectants (avoid repeated freeze-thaw cycles)

• For RNA-binding studies: Include RNase inhibitors and DEPC-treated solutions

What methodological approaches are most effective for studying rplR-RNA interactions in D. psychrophila?

For investigating the interactions between D. psychrophila rplR and its RNA partners:

• Electrophoretic Mobility Shift Assays (EMSA):

  • Prepare labeled 5S rRNA and 23S rRNA fragments

  • Incubate with purified recombinant rplR at 4°C

  • Run native gels at 4°C to maintain complex integrity

  • Include competitions with unlabeled RNA to determine specificity

• RNA footprinting analysis:

  • Use chemical (DMS, CMCT) or enzymatic (RNase) probing

  • Map protection patterns when rplR is bound

  • Compare footprints at different temperatures (0-25°C)

  • Utilize next-generation sequencing for high-resolution mapping

• Fluorescence-based approaches:

  • Label rplR with fluorescent probes minimally affecting function

  • Measure binding kinetics through fluorescence anisotropy

  • Use FRET to determine conformational changes upon binding

  • Perform assays at multiple temperatures to establish thermodynamic parameters

• Quantitative binding studies:

  • Determine association/dissociation constants using surface plasmon resonance

  • Compare binding energetics with mesophilic L18 proteins

  • Analyze enthalpy-entropy compensation mechanisms at different temperatures

  • Include Mg²⁺ titrations to assess cation dependence of interactions

• In vitro reconstitution:

  • Assemble partial ribosomal complexes with rplR and partner proteins (e.g., L5)

  • Assess integration with 5S rRNA at low temperatures

  • Use cryo-electron microscopy to visualize complex formation

  • Compare assembly efficiency with mesophilic components
    When designing these experiments, researchers should include appropriate controls for non-specific binding and ensure all equipment is calibrated for reliable low-temperature measurements.

How should researchers approach mutagenesis studies to identify cold-adaptation determinants in D. psychrophila rplR?

A systematic mutagenesis approach would involve:

• Comparative sequence analysis:

  • Align D. psychrophila rplR with homologs from mesophilic and thermophilic bacteria

  • Identify residues unique to psychrophilic species

  • Map these residues onto structural models to identify potential adaptation sites

  • Focus on surface-exposed residues and those involved in RNA binding

• Targeted mutagenesis strategy:

  • Design substitutions converting psychrophilic-specific residues to mesophilic equivalents

  • Create complementary mutations introducing psychrophilic features into mesophilic proteins

  • Generate conservative and non-conservative substitutions at key positions

  • Develop a library of chimeric proteins with domain swaps between psychrophilic and mesophilic L18s

• Functional assays for mutant proteins:

  • Measure thermal stability using differential scanning calorimetry

  • Assess RNA binding capacity at various temperatures

  • Test ability to incorporate into ribosomal subunits

  • Evaluate translation activity in reconstituted systems

  • Perform complementation tests in conditional knockout strains

• Data analysis approach:

  • Correlate changes in stability/activity with specific mutations

  • Develop statistical models relating sequence features to cold adaptation

  • Use machine learning to identify patterns across multiple mutations

  • Create structure-function relationship maps This methodological framework will enable researchers to systematically identify the molecular determinants that allow D. psychrophila rplR to function optimally in cold environments, providing insights into evolutionary adaptation mechanisms.