Recombinant Photobacterium profundum 50S ribosomal protein L15 (rplO)

What is the structural role of ribosomal protein L15 in Photobacterium profundum's 50S subunit?

Ribosomal protein L15 in P. profundum, like its homologs in other bacteria, plays a critical role in the assembly and structural integrity of the 50S ribosomal subunit. Based on studies in other bacterial systems, L15 interacts with over ten other proteins during 50S assembly in vitro . It serves as a key binding protein that helps establish the tertiary structure of the large subunit through specific interactions with 23S rRNA.

Methodological approach for studying L15 structural interactions:

• Isolate core particles derived from P. profundum 50S subunits using LiCl treatment (typically 2M concentration)

• Purify recombinant L15 protein expressed in a heterologous system

• Reconstitute purified L15 with core particles

• Perform chemical footprinting using Fe(II)-EDTA and dimethyl sulfate

• Analyze the region of interaction on 23S rRNA (likely focusing on domains II, I, IV, and V)

In E. coli, a model organism for ribosomal studies, L15 has a strong footprint in the region spanning nucleotides 572-654 in domain II of 23S rRNA . This interaction pattern provides a starting reference for investigating P. profundum L15's binding sites.

How does growth pressure affect the expression and function of L15 in P. profundum?

As a piezophilic bacterium, P. profundum grows optimally at 28 MPa and 15°C , suggesting its cellular machinery, including ribosomal proteins, has evolved specific adaptations for high-pressure environments. While the specific pressure effects on L15 expression haven't been directly documented in the available literature, we can extrapolate from pressure-adaptation studies in P. profundum.

Protocol for analyzing pressure-dependent L15 expression:

• Culture P. profundum at varying pressures (atmospheric, 15 MPa, 28 MPa, 40 MPa)

• Extract total cellular protein

• Perform quantitative proteomics analysis using MS-based label-free methods

• Compare L15 expression levels across pressure conditions

• Conduct parallel transcriptomic analysis to determine if regulation occurs at the mRNA level

Pressure-related adaptations in ribosomal proteins typically include modified amino acid compositions and altered protein-RNA interaction surfaces that maintain functionality under compression.

What experimental approaches are recommended for purifying recombinant P. profundum L15?

Based on protocols for similar ribosomal proteins, the following methodology is recommended:

Table 1: Purification Protocol for Recombinant P. profundum L15

This protocol is adaptable based on requirements for downstream applications such as structural studies or functional assays.

How can chemical footprinting be optimized to study L15-rRNA interactions in P. profundum under high-pressure conditions?

Standard chemical footprinting techniques require significant modifications for high-pressure applications with P. profundum L15. Based on previously established techniques for E. coli L15 , the following optimized protocol can be implemented:

• Pressure-adapted reaction chamber design:

  • Use specialized high-pressure vessels capable of maintaining 28 MPa during chemical reactions

  • Incorporate remote injection systems for reagents to initiate reactions under pressure

  • Include temperature control to maintain optimal 15°C

• Modified footprinting reagents:

  • For hydroxyl radical probing, use Fe(II)-EDTA with rapid mixing mechanisms

  • For base-specific probing, use dimethyl sulfate with pressure-stable carriers

  • Test pressure effects on chemical reaction rates and adjust exposure times accordingly

• Control experiments required:

  • Parallel footprinting at atmospheric pressure as baseline

  • Naked 23S rRNA controls under identical pressure conditions

  • Comparative footprinting with E. coli L15 under high pressure

The footprinting patterns would likely reveal pressure-specific conformational changes in the L15-rRNA interaction interface, particularly in the domain II region (nucleotides 572-654) known to interact with L15 in E. coli .

What role does L15 play in the assembly pathway of the 50S subunit in P. profundum, and how does pressure affect this process?

The assembly of 50S ribosomal subunits in P. profundum likely follows a pressure-optimized pathway in which L15 serves as a critical assembly factor. Based on ribosomal assembly studies in other bacteria and research on ribosomal protein functions:

Table 2: Predicted Assembly Pathway Steps Involving L15 in P. profundum

To experimentally validate this pathway:

• Generate partially reconstituted particles at different assembly stages

• Use sucrose gradient analysis to monitor the conversion of pre-ribosomal particles to mature 50S subunits

• Compare assembly rates and intermediates at different pressures

• Use depletion studies to assess how L15 absence affects the formation of specific assembly intermediates

Knockdown experiments with L15 would likely show significant reduction in pre-60S ribosomal subunits similar to that observed with other essential ribosomal proteins .

How can protein-tethered hydroxyl radical probing be adapted to map the three-dimensional environment of L15 in P. profundum ribosomes?

Based on the methodology described for E. coli L15 , the following protocol can be adapted for P. profundum:

• Generation of single-cysteine L15 mutants:

  • Create recombinant P. profundum L15 variants with single cysteine residues at strategic positions (similar to positions 68, 71, and 115 used in E. coli studies)

  • Express and purify these variants under conditions that maintain native folding

• Derivatization with Fe(II)-EDTA:

  • React the cysteine residues with 1-[p-(bromoacetamido)benzyl]-EDTA

  • Chelate with Fe(II) to create localized hydroxyl radical generators

  • Verify derivatization by mass spectrometry

• Reconstitution and radical generation:

  • Incorporate derivatized L15 into core particles derived from P. profundum 50S subunits

  • Initiate hydroxyl radical production in pressure vessels

  • Terminate reactions rapidly to prevent secondary damage

• Analysis of cleavage patterns:

  • Extract and analyze 23S rRNA cleavage sites using primer extension or next-generation sequencing

  • Map cleavage sites onto secondary structure models

  • Generate 3D interaction maps based on cleavage intensity and distance constraints

This approach would reveal specific rRNA elements that are in close proximity to L15 in the assembled ribosome, providing constraints on the tertiary folding of 23S rRNA under pressure conditions.

What adaptations in P. profundum L15 enable ribosome function at high hydrostatic pressure?

While specific adaptations in P. profundum L15 have not been fully characterized, likely adaptations can be inferred from general principles of protein adaptation to high pressure environments:

Table 3: Predicted Pressure Adaptations in P. profundum L15

To experimentally validate these adaptations:

• Perform comparative structural analysis of P. profundum L15 versus mesophilic homologs

• Conduct site-directed mutagenesis targeting predicted pressure-adaptive features

• Measure binding kinetics and thermodynamics under varying pressure conditions

• Analyze the effects of heterologous expression (P. profundum L15 in E. coli and vice versa) on pressure tolerance

How can mutational analysis of the rplO gene inform our understanding of P. profundum's piezoadaptation?

Systematic mutational analysis of the P. profundum rplO gene would provide valuable insights into the molecular basis of pressure adaptation. Based on approaches used for studying other pressure-adaptive genes in P. profundum :

• Generation of rplO mutant library:

  • Create point mutations, deletions, and domain swaps with non-piezophilic homologs

  • Introduce these mutations into P. profundum using conjugation systems

  • Verify mutations by sequencing

• Phenotypic screening under pressure:

  • Assess growth rates across a pressure gradient (atmospheric to 40 MPa)

  • Analyze ribosome assembly efficiency using sucrose gradient centrifugation

  • Measure translation rates using reporter constructs

• Complementation studies:

  • Test whether wild-type rplO can restore normal growth in mutant strains

  • Introduce P. profundum rplO into E. coli and assess pressure tolerance

  • Create chimeric L15 proteins to identify critical domains

• Structural validation:

  • Determine structures of wild-type and mutant L15 proteins

  • Map mutations onto structural models to correlate with functional effects

This approach parallels methods used to study the pressure-adaptive role of RecD in P. profundum, where gene disruption created pressure-sensitive phenotypes and complementation studies confirmed gene function .

What are the key considerations for designing expression systems for P. profundum ribosomal proteins?

When designing expression systems for P. profundum L15 and other ribosomal proteins, several factors must be considered:

• Codon optimization:

  • P. profundum has a GC content of approximately 50%

  • Optimize codons for the expression host while maintaining critical features

  • Consider the impact of rare codons on translation efficiency

• Expression host selection:

  • E. coli-based systems offer simplicity but may not provide optimal folding

  • Yeast expression systems have been successful for other P. profundum ribosomal proteins

  • Cold-adapted expression hosts may improve folding at lower temperatures

• Induction and growth conditions:

  • Low-temperature induction (15-20°C) mimics P. profundum's natural environment

  • Extended expression periods (24-48 hours) often yield better results

  • Consider using auto-induction media to avoid rapid protein accumulation

• Protein solubility enhancement:

  • Fusion partners (MBP, SUMO, etc.) can improve solubility

  • Co-expression with chaperones may facilitate proper folding

  • Addition of osmolytes or pressure treatment during expression may enhance native folding

What analytical techniques are most suitable for functional assessment of recombinant P. profundum L15?

To fully characterize the functionality of recombinant P. profundum L15, multiple analytical approaches should be employed:

Table 4: Analytical Techniques for P. profundum L15 Functional Assessment

When conducting these analyses, it is critical to compare results at both atmospheric pressure and the optimal growth pressure of P. profundum (28 MPa) to identify pressure-specific functional characteristics.

What are the most promising research directions for understanding P. profundum L15's role in piezoadaptation?

Based on current knowledge gaps and research trends, the following directions represent high-priority areas for advancing our understanding of P. profundum L15:

• Comprehensive comparative structural analysis:

  • Determine high-resolution structures of P. profundum L15 under varying pressure conditions

  • Compare with homologs from non-piezophilic bacteria to identify adaptation signatures

  • Develop computational models to predict pressure effects on ribosomal protein function

• In vivo ribosome assembly studies:

  • Develop methods to track ribosome assembly in living P. profundum cells under pressure

  • Use fluorescently tagged L15 to monitor its incorporation into ribosomes in real-time

  • Identify pressure-specific assembly intermediates and pathways

• Systems biology approach:

  • Integrate proteomic, transcriptomic, and structural data to build comprehensive models

  • Analyze co-evolution patterns between L15 and other ribosomal components

  • Identify regulatory networks controlling L15 expression under pressure stress

• Synthetic biology applications:

  • Engineer pressure-adapted ribosomes containing P. profundum L15 for biotechnological applications

  • Develop pressure-resistant protein synthesis systems for industrial processes

  • Create chimeric ribosomes with enhanced functionality under extreme conditions

These research directions would significantly advance our understanding of ribosomal adaptation to extreme environments while potentially yielding biotechnological applications for high-pressure bioprocessing.