Recombinant Prochlorococcus marinus subsp. pastoris 50S ribosomal protein L35 (rpmI)

What is the biological significance of the 50S ribosomal protein L35 in Prochlorococcus marinus subsp. pastoris?

The 50S ribosomal protein L35 (rpmI) in Prochlorococcus marinus subsp. pastoris (CCMP1986/MED4) is a critical component of the large ribosomal subunit involved in protein synthesis. This protein plays an essential role in the organism's translation machinery, which is particularly interesting given that P. marinus has one of the smallest genomes among photosynthetic organisms - a single circular chromosome of only 1,657,990 bp containing 1,796 predicted protein-coding genes . The ribosomal proteins in this minimalist genome have been under strong selective pressure to maintain core cellular functions while shedding non-essential features, making them valuable models for studying molecular evolution and adaptation to oligotrophic environments.

Research methodology for investigating L35 significance includes:

• Comparative genomic analysis across Prochlorococcus ecotypes

• Structural alignment with L35 proteins from other cyanobacteria

• Ribosome profiling to examine translation efficiency

• Site-directed mutagenesis to assess functional domains

How does the amino acid sequence of Prochlorococcus marinus L35 differ from other marine cyanobacteria?

Prochlorococcus marinus strains have undergone genome streamlining while maintaining essential functions. The 50S ribosomal protein L35, as a core component of the translation machinery, exhibits sequence conservation in functional domains while showing divergence in non-critical regions when compared to other marine cyanobacteria.

Methodological approach for sequence analysis:

• Multiple sequence alignment using MUSCLE or CLUSTAL programs

• Calculation of sequence identity and similarity percentages

• Identification of conserved motifs using MEME Suite

• Phylogenetic analysis using maximum likelihood methods

Typical sequence conservation patterns observed:

While the specific sequence of the Prochlorococcus marinus subsp. pastoris L35 protein is not fully detailed in the search results, recombinant versions are available commercially for research purposes .

What are the optimal expression systems for producing recombinant Prochlorococcus marinus subsp. pastoris 50S ribosomal protein L35?

Based on available commercial products, E. coli is the predominant expression system for recombinant Prochlorococcus marinus 50S ribosomal protein L35 (rpmI) . The methodological approach for optimizing expression includes:

• Vector selection:

  • pET series vectors with T7 promoter systems

  • pBAD vectors for arabinose-inducible expression

  • pCold vectors for cold-shock induced expression

• E. coli strain optimization:

  • BL21(DE3) for standard expression

  • Rosetta for rare codon optimization

  • Arctic Express for improved protein folding at lower temperatures

• Expression conditions optimization matrix:

• Solubility enhancement strategies:

  • Fusion tags (His, GST, MBP, SUMO)

  • Co-expression with chaperones

  • Addition of solubility enhancers to media

Commercial recombinant versions typically achieve >85% purity as assessed by SDS-PAGE , providing researchers with reliable starting material for structural and functional studies.

What purification strategies yield the highest activity for recombinant Prochlorococcus marinus L35 protein?

Multi-step purification approaches are recommended to obtain highly pure, biologically active recombinant L35 protein:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • Glutathione affinity for GST-fusion proteins

  • Amylose resin for MBP-fusion proteins

• Intermediate purification:

  • Ion exchange chromatography based on the protein's theoretical pI

  • Hydrophobic interaction chromatography

• Polishing step:

  • Size exclusion chromatography to remove aggregates and obtain monodisperse protein

• Quality control assessment:

  • SDS-PAGE (target: >95% purity)

  • Western blot confirmation

  • Mass spectrometry verification

  • Dynamic light scattering for monodispersity

  • Circular dichroism for secondary structure confirmation

• Storage optimization:

  • Recommended storage in buffer containing 20-50% glycerol at -20°C/-80°C

  • Aliquoting to prevent repeated freeze-thaw cycles

  • Addition of reducing agents if cysteine residues are present

According to product specifications, the shelf life of liquid recombinant protein preparations is typically 6 months at -20°C/-80°C, while lyophilized forms can remain stable for up to 12 months .

What methods are most effective for studying Prochlorococcus marinus L35 interactions with ribosomal RNA?

Investigating the interactions between the 50S ribosomal protein L35 and ribosomal RNA requires a combination of biophysical and biochemical approaches:

These methodologies can help elucidate how the minimal genome of Prochlorococcus marinus (1,657,990 bp with only 1,796 protein-coding genes) has maintained efficient translation machinery despite extreme genome streamlining.

How can researchers resolve contradictory structural data for the L35 protein across different Prochlorococcus ecotypes?

When faced with contradictory structural data for L35 across different Prochlorococcus ecotypes, researchers should implement a systematic approach to resolve discrepancies:

• Standardization of experimental conditions:

  • Consistent buffer compositions and pH

  • Uniform protein preparation methods

  • Identical structural determination protocols

• Multi-method validation approach:

  • Compare results from X-ray crystallography, NMR, and cryo-EM

  • Use computational structure prediction as an independent verification

  • Apply hydrogen-deuterium exchange mass spectrometry to assess conformational dynamics

• Ecotype-specific variation analysis:

  • Create a systematized table comparing key structural parameters across ecotypes

  • Correlate structural differences with adaptive phenotypes

  • Consider the impact of high-light vs. low-light adaptation on protein structure

• Resolution of contradictions methodology:

  • Identify whether contradictions are due to technical artifacts or biological variation

  • Perform directed evolution experiments to test functional implications of structural differences

  • Use ancestral sequence reconstruction to trace the evolutionary trajectory of structural changes

The high genetic diversity observed among Prochlorococcus strains (with a pangenome containing more than 80,000 genes despite their small individual genomes) suggests that structural variations in ribosomal proteins might contribute to niche adaptation in different oceanic environments.

How can recombinant L35 protein be used to study the evolutionary adaptation of Prochlorococcus to different ocean environments?

Recombinant L35 protein serves as an excellent model for studying evolutionary adaptation of Prochlorococcus through several methodological approaches:

• Comparative biochemical characterization:

  • Measure thermal stability across L35 variants from different oceanic regions

  • Assess pH optimum differences in protein function

  • Compare salt tolerance profiles using stability assays

• Reconstituted translation systems:

  • Create hybrid ribosomes with components from different ecotypes

  • Measure translation efficiency under various environmental conditions

  • Quantify error rates and fidelity across temperature gradients

• Molecular evolution analysis:

  • Calculate Ka/Ks ratios to detect selection signatures

  • Reconstruct ancestral sequences to trace evolutionary trajectories

  • Identify coevolving residues between L35 and interacting partners

• Environmental adaptation experiments:

  • Express L35 variants in model organisms under stress conditions

  • Measure fitness effects through growth assays

  • Use directed evolution to identify adaptive mutations

This research is particularly relevant given that Prochlorococcus contributes 30-80% of total photosynthesis in oligotrophic oceans, playing a significant role in the global carbon cycle . The evolutionary adaptations of its ribosomal components may help explain its ecological success across diverse marine environments.

What genomic evidence suggests selection pressure on the rpmI gene in Prochlorococcus marinus evolution?

Several lines of genomic evidence point to selection pressure on the rpmI gene encoding the 50S ribosomal protein L35:

• Sequence conservation analysis:

  • Core functional domains show strong negative selection

  • Surface-exposed residues exhibit higher variability

  • RNA-binding motifs maintain higher conservation than protein-protein interaction sites

• Codon usage patterns:

  • Bias toward optimal codons in high-expression ribosomal genes

  • Correlation between codon adaptation index and expression levels

  • Methodological approach: Calculate relative synonymous codon usage (RSCU) values

• Comparative genomic evidence:

  • Synteny analysis of the genomic neighborhood around rpmI

  • Presence/absence of regulatory elements across strains

  • Association with genomic islands or horizontally transferred regions

• Molecular evolution signatures:

The compact genome of Prochlorococcus marinus subsp. pastoris CCMP1986 (MED4), with only 1,657,990 bp , suggests strong selective pressure for genome streamlining while maintaining essential functions like translation, which would directly impact ribosomal proteins like L35.

How can cryo-electron microscopy be optimized to study Prochlorococcus ribosomes containing recombinant L35 protein?

Optimizing cryo-electron microscopy (cryo-EM) for studying Prochlorococcus ribosomes with recombinant L35 requires specialized methodological approaches:

• Sample preparation optimization:

  • Ribosome isolation through sucrose gradient ultracentrifugation

  • Buffer screening for optimal particle distribution

  • Grid optimization with different carbon supports and hole sizes

  • Controlled denaturation experiments to assess L35 contribution to stability

• Data collection strategy:

  • Use of energy filters to improve signal-to-noise ratio

  • Phase plate implementation for enhanced contrast

  • Motion correction with dose fractionation

  • Tilt series acquisition for dealing with preferred orientation

• Image processing workflow:

  • 2D classification to identify intact ribosomes

  • Ab initio model generation without reference bias

  • Focused refinement on the L35 region

  • Local resolution estimation to identify dynamic regions

• Validation and interpretation:

  • Confirmation with recombinant L35 labeled with gold nanoparticles

  • Integration with crosslinking mass spectrometry data

  • Molecular dynamics flexible fitting to interpret conformational states

  • Comparison with ribosomes from different Prochlorococcus ecotypes

This approach allows researchers to understand how the unique adaptations of Prochlorococcus ribosomes, including the role of L35, contribute to the remarkable success of this organism that dominates the temperate and tropical oceans .

What techniques can effectively measure the impact of L35 variants on ribosome assembly and translation efficiency?

A multi-faceted approach combining biochemical, biophysical, and computational methods provides the most comprehensive assessment of L35 variant effects:

These methodologies can help understand how Prochlorococcus maintains efficient translation machinery despite its minimal genome, which might contribute to its ecological success as the most abundant photosynthetic organism in many oceanic regions .

What are the most common technical challenges when working with recombinant Prochlorococcus marinus L35 protein and how can they be overcome?

Researchers frequently encounter several challenges when working with recombinant Prochlorococcus marinus L35 protein:

• Solubility issues:

  • Challenge: L35 may form inclusion bodies in E. coli expression systems

  • Solutions:

    • Lower expression temperature (16-20°C)

    • Use solubility-enhancing fusion tags (MBP, SUMO)

    • Add solubility enhancers to lysis buffer (non-detergent sulfobetaines, arginine)

    • Optimize codon usage for E. coli expression

• Stability challenges:

  • Challenge: Protein degradation during purification or storage

  • Solutions:

    • Add protease inhibitors during purification

    • Include reducing agents to prevent disulfide formation

    • Store in buffer with 20-50% glycerol at -80°C

    • Avoid repeated freeze-thaw cycles as recommended in product specifications

• RNA contamination:

  • Challenge: Co-purification of bacterial RNA due to L35's RNA-binding properties

  • Solutions:

    • Include RNase treatment during purification

    • Use high-salt washes during affinity chromatography

    • Apply polyethyleneimine precipitation to remove nucleic acids

    • Implement additional ion-exchange chromatography steps

• Functional assay development:

  • Challenge: Demonstrating that recombinant L35 retains native functionality

  • Solutions:

    • Develop RNA binding assays with synthetic rRNA fragments

    • Establish reconstitution assays with other ribosomal components

    • Use circular dichroism to confirm proper folding

    • Implement thermal shift assays to verify ligand binding

A systematic approach to troubleshooting these issues will significantly improve research outcomes when working with this challenging but important protein.

How can researchers address contradictory results in experimental studies of L35 function across different Prochlorococcus ecotypes?

When faced with contradictory results regarding L35 function across different Prochlorococcus ecotypes, researchers should implement a systematic resolution strategy:

• Standardization of experimental conditions:

  • Establish a controlled comparison framework:

  Variable	Standardization approach	Monitoring method
  Protein preparation	Identical purification protocols	SDS-PAGE, western blot
  Buffer conditions	Matched pH, salt, and additives	Conductivity, pH measurement
  Assay temperature	Conduct assays at multiple defined temperatures	Temperature logs
  RNA substrates	Standardized in vitro transcription	Gel electrophoresis

• Cross-validation with multiple techniques:

  • Apply complementary methodologies to verify results:

    • Both in vitro and in vivo functional assays

    • Direct biochemical measurements and genetic approaches

    • Structural studies and computational predictions

    • Evolutionary analyses and experimental evolution

• Ecological context integration:

  • Consider the natural environment of different ecotypes:

    • Test function across temperature ranges matching oceanic distributions

    • Examine performance under relevant light conditions

    • Assess salt and pH tolerances reflecting natural habitats

    • Evaluate performance under nutrient limitations typical of oligotrophic environments

• Collaborative validation:

  • Implement inter-laboratory validation studies:

    • Share reagents, protocols, and samples between research groups

    • Blind testing of samples to reduce experimental bias

    • Develop standardized reporting formats for L35 functional data

    • Establish a database of experimental conditions and results

This systematic approach recognizes that Prochlorococcus strains are highly diverse with a pangenome containing more than 80,000 genes , and functional differences in L35 may reflect genuine adaptations to different oceanic niches rather than experimental artifacts.

What emerging technologies will advance our understanding of Prochlorococcus ribosomal proteins like L35?

Several cutting-edge technologies are poised to transform research on Prochlorococcus ribosomal proteins:

• Advanced structural biology approaches:

  • Cryo-electron tomography for in situ visualization of ribosomes in Prochlorococcus cells

  • Integrative structural biology combining cryo-EM, cross-linking mass spectrometry, and computational modeling

  • Time-resolved X-ray free-electron laser studies to capture ribosome dynamics

  • Microcrystal electron diffraction for structural analysis of challenging ribosomal proteins

• Single-molecule techniques:

  • Optical tweezers to measure forces during translation

  • Single-molecule FRET to observe conformational changes in L35 during ribosome function

  • Nanopore sensing for detecting ribosomal protein-RNA interactions

  • Zero-mode waveguides for real-time observation of translation

• Systems biology approaches:

  • Ribosome profiling across environmental gradients to link L35 function to ecological adaptation

  • Multi-omics integration (transcriptomics, proteomics, metabolomics) to understand system-level effects

  • Mathematical modeling of translation efficiency in minimal genomes

  • Flux balance analysis incorporating translation constraints

• Synthetic biology tools:

  • Genome editing with CRISPR-Cas9 for targeted modification of ribosomal proteins

  • Minimal synthetic ribosomes incorporating only essential components

  • Cell-free translation systems with defined components for mechanistic studies

  • Expansion of the genetic code to incorporate non-canonical amino acids into L35 for functional studies

These technologies will help reveal how the minimal genome of Prochlorococcus (1,657,990 bp with 1,796 protein-coding genes) maintains efficient translation machinery that supports its ecological dominance in oligotrophic oceans.

How might climate change impact the evolution of ribosomal proteins like L35 in marine cyanobacteria?

Climate change may drive significant evolutionary pressure on ribosomal proteins like L35 in Prochlorococcus, with several research approaches available to investigate this phenomenon:

• Experimental evolution studies:

  • Long-term cultivation under projected future ocean conditions:

    • Elevated temperatures (warming scenarios)

    • Decreased pH (ocean acidification)

    • Altered nutrient availability

    • Changed light penetration due to stratification

  • Whole-genome sequencing to identify adaptive mutations

  • Focused analysis of ribosomal protein genes including rpmI (L35)

• Comparative genomics across environmental gradients:

  • Sampling Prochlorococcus populations across:

    • Latitude gradients (temperature proxies)

    • Depth gradients (light and temperature proxies)

    • Seasonal cycles (temporal environmental variation)

  • Targeted sequencing of ribosomal protein genes

  • Analysis of selection signatures in contemporary populations

• Molecular function assessment under climate stress:

  • Thermal stability profiling of L35 variants

  • pH-dependent ribosome assembly and function analysis

  • Protein-RNA interaction strength under varying conditions

  • Translation efficiency and accuracy measurements

• Predictive modeling approaches:

  • Molecular dynamics simulations under altered environmental parameters

  • Population genetic modeling of selection scenarios

  • Integration with ocean circulation and biogeochemical models

  • Machine learning approaches to identify climate-sensitive residues in L35