Recombinant Idiomarina loihiensis 50S ribosomal protein L35 (rpmI)

What is the L35 ribosomal protein and what is its role in bacterial ribosomes?

The L35 (rpmI) protein is a component of the 50S ribosomal subunit in bacteria. It plays a crucial role in ribosome assembly and stability. Based on research with other ribosomal proteins, L35 likely participates in the formation of functional ribosomes by binding to specific regions of rRNA and facilitating proper folding of the ribosomal structure . In bacteria, L35 is typically involved in late-stage assembly of the 50S subunit and may contribute to the association of the 50S and 30S subunits to form the complete 70S ribosome.

How does L35 in Idiomarina loihiensis compare to homologous proteins in other bacteria?

The L35 protein in I. loihiensis likely shares structural similarities with homologs in other γ-proteobacteria, though with specific adaptations to the extreme deep-sea environment. While complete characterization of I. loihiensis L35 is still ongoing, comparative genomic analysis has shown that most I. loihiensis proteins have closest homologs in other γ-proteobacteria (approximately 77%) . Like other ribosomal proteins, L35 is likely highly conserved but may contain unique adaptations that contribute to ribosome stability under high pressure and fluctuating temperature conditions characteristic of hydrothermal vents.

Where is the rpmI gene located in the I. loihiensis genome?

The rpmI gene encoding L35 is located within the 2,839,318 bp circular chromosome of I. loihiensis . As with many bacterial genomes, ribosomal protein genes are often organized in operons. In I. loihiensis, the genome contains four rRNA operons and 56 tRNA genes, accounting for part of the 92.1% coding capacity of the genome . The exact position of rpmI would need to be determined by specific genomic analysis, but it is likely part of a ribosomal protein operon, as is common in prokaryotes.

What expression systems are most effective for recombinant I. loihiensis L35 production?

For recombinant production of I. loihiensis L35 protein, E. coli-based expression systems are typically most effective. Based on protocols similar to those used for other ribosomal proteins, such as the L10 protein from I. loihiensis , the gene should be cloned into an expression vector with an appropriate promoter (such as T7) and affinity tag (such as His-tag or GST-tag). BL21(DE3) or similar E. coli strains are recommended hosts for expression. The expression conditions should be optimized for temperature (typically 16-30°C), induction time (4-16 hours), and IPTG concentration (0.1-1.0 mM) to balance protein yield and solubility.

What purification strategies yield the highest purity of recombinant L35 from I. loihiensis?

A multi-step purification strategy is recommended for obtaining high-purity L35 protein. Based on standard protocols for ribosomal proteins, this typically includes:

• Affinity chromatography: Using a His-tag system with Ni-NTA resin if the protein is expressed with a polyhistidine tag

• Ion-exchange chromatography: To separate based on charge differences

• Size-exclusion chromatography: For final polishing and buffer exchange

The target purity should be >85% as assessed by SDS-PAGE, similar to the standards for other recombinant ribosomal proteins like L10 . Buffer conditions should be optimized to maintain protein stability, typically including 20-50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, and potentially 5-10% glycerol to improve stability during storage at -20°C or -80°C.

How can researchers assess the proper folding and activity of recombinant L35?

The proper folding of recombinant L35 can be assessed through multiple complementary approaches:

• Circular dichroism (CD) spectroscopy to evaluate secondary structure content

• Thermal shift assays to determine protein stability

• RNA binding assays to confirm functional activity, as L35 should bind to specific rRNA sequences

• In vitro ribosome assembly assays to test the protein's ability to incorporate into partial or complete ribosomal structures

The gold standard would be demonstrating that the recombinant L35 can complement L35-depleted ribosomes or cells, similar to studies performed with L35 in other organisms .

How can researchers study the role of L35 in ribosome assembly specific to I. loihiensis?

To study L35's role in I. loihiensis ribosome assembly, researchers should consider:

• In vitro reconstitution assays: Using purified components to reconstruct partial or complete ribosomal subunits

• Cryo-electron microscopy (cryo-EM): To visualize the structural position of L35 within the assembled ribosome

• Depletion studies: Creating conditional mutants where L35 expression can be regulated to observe effects on ribosome assembly

• Pulse-chase analyses: To track the kinetics of ribosome assembly in the presence and absence of functional L35

• Northern hybridization and primer extension analyses: To monitor pre-rRNA processing defects that may occur upon L35 depletion or mutation, similar to techniques used in yeast studies

These approaches would help determine if I. loihiensis L35, like its counterparts in other organisms, is critical for specific steps in ribosome maturation.

What techniques are most appropriate for studying L35 interactions with rRNA and other ribosomal proteins?

Several complementary techniques can effectively characterize L35 interactions:

• RNA electrophoretic mobility shift assays (EMSA): To detect direct binding between purified L35 and specific rRNA fragments

• Surface plasmon resonance (SPR): To measure binding kinetics and affinity

• Cross-linking followed by mass spectrometry: To identify contact sites between L35 and other ribosomal components

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS): To map interaction surfaces

• Yeast two-hybrid or bacterial two-hybrid systems: To screen for protein-protein interactions

• Co-immunoprecipitation: To pull down L35 along with its interaction partners from cell lysates

These methods, when used in combination, can provide a comprehensive map of L35's interaction network within the ribosome.

How can the effect of extreme conditions on L35 function be assessed experimentally?

Given I. loihiensis' adaptation to extreme deep-sea conditions, studying L35 function under such conditions is particularly relevant:

• Thermal stability assays: Compare the stability of I. loihiensis L35 with homologs from mesophilic bacteria across a temperature range (4-50°C)

• Pressure chamber experiments: Evaluate protein folding and RNA binding under high hydrostatic pressure (up to 130 atmospheres, similar to its native environment)

• Salt tolerance assays: Test functional activity in buffers with varying NaCl concentrations (0.5-20%)

• Comparative ribosome assembly assays: Conduct assembly experiments under varying conditions to identify environment-specific adaptations

• Molecular dynamics simulations: Model the behavior of L35 under different temperature and pressure conditions

These experiments would help identify unique adaptations that allow I. loihiensis L35 to function in its extreme native environment.

How does L35 from I. loihiensis compare structurally and functionally to L35 from other extremophiles?

Comparative analysis of L35 from I. loihiensis and other extremophiles would reveal evolutionary adaptations to extreme environments:

• Sequence alignment analysis: Identify conserved and variable regions across extremophiles

• 3D structure comparisons: If structures are available, analyze differences in folding, surface charge distribution, and hydrophobic core

• Thermostability measurements: Compare melting temperatures of L35 proteins from different extremophiles

• Functional complementation: Test whether L35 from different extremophiles can functionally substitute for each other

• Molecular phylogenetic analysis: Construct evolutionary trees to understand the relationship between L35 variants

This comparative approach would highlight specific adaptations that have evolved in response to different extreme environments.

What insights can comparison of I. loihiensis L35 with homologs from non-extremophiles provide?

Comparing I. loihiensis L35 with homologs from non-extremophiles can reveal:

• Structural adaptations: Specific amino acid substitutions that confer stability under extreme conditions

• Functional differences: Variations in binding affinities, assembly kinetics, or rRNA processing roles

• Evolutionary rate: Whether L35 in extremophiles evolves faster or slower than in mesophiles

• Selective pressures: Identification of residues under positive selection that may contribute to environmental adaptation

• Functional plasticity: The degree to which L35 function is conserved despite sequence divergence

Such comparisons would contribute to understanding how core cellular machinery adapts to extreme environments while maintaining essential functions.

How can researchers use I. loihiensis L35 to study ribosome adaptation to extreme environments?

I. loihiensis L35 offers a unique system for studying ribosomal adaptation:

• Chimeric ribosomes: Create hybrid ribosomes with L35 from I. loihiensis and other components from mesophilic bacteria to identify specific adaptations

• Site-directed mutagenesis: Systematically alter key residues to determine their contribution to environmental adaptation

• In vitro translation assays: Compare translation efficiency and accuracy under various conditions

• Ribosome profiling: Assess whether L35 variants affect ribosome pausing or codon usage preferences

• Structural studies: Use cryo-EM or crystallography to visualize how L35 contributes to ribosome stability under extreme conditions

These approaches would help understand how ribosomes, among the most conserved cellular machines, adapt to function in extreme environments.

What is the potential role of L35 in pre-rRNA processing in I. loihiensis compared to other organisms?

Based on studies in yeast, L35 plays a critical role in pre-rRNA processing . In I. loihiensis, this role may be conserved but with specific adaptations:

• rRNA processing pathway analysis: Map the pre-rRNA processing pathway in I. loihiensis using northern blot and primer extension

• L35 depletion studies: Analyze effects on specific processing steps when L35 is absent or mutated

• Binding site identification: Determine L35 binding sites on pre-rRNA using CRAC (crosslinking and analysis of cDNA) or similar techniques

• Interactome studies: Identify processing factors that interact with L35 using pull-down assays followed by mass spectrometry

• Comparative analysis: Compare processing defects in L35-depleted I. loihiensis with those observed in other organisms like yeast

This research would clarify whether the role of L35 in pre-rRNA processing is universal or has evolved specific features in extremophiles.

How might I. loihiensis L35 be utilized in structural biology studies of extremophile ribosomes?

I. loihiensis L35 can contribute significantly to structural biology studies:

• High-resolution structures: Obtain crystal or cryo-EM structures of I. loihiensis ribosomes, focusing on L35's position and interactions

• Conformational dynamics: Use single-molecule FRET to study dynamic aspects of L35 within the ribosome under varying conditions

• Comparative structural biology: Analyze structural differences between I. loihiensis ribosomes and those from mesophiles

• Structure-based design: Engineer ribosomes with enhanced stability based on insights from I. loihiensis L35

• In silico molecular dynamics: Model the behavior of L35 within the ribosome under different temperature and pressure conditions

These structural studies would improve our understanding of how ribosomes maintain functionality in extreme environments.

What are common challenges in expressing recombinant I. loihiensis L35 and how can they be addressed?

Researchers commonly encounter several challenges when expressing recombinant ribosomal proteins:

• Protein solubility issues: Address by optimizing expression temperature (typically lowering to 16-18°C), using solubility-enhancing tags, or adding chemical chaperones

• Protein toxicity to host cells: Use tightly regulated expression systems and consider codon optimization for the host organism

• Incorrect folding: Incorporate molecular chaperones (GroEL/GroES) in the expression system

• Degradation during purification: Add protease inhibitors and work at 4°C during all purification steps

• Low yield: Optimize induction conditions and consider using specialized strains like Rosetta for rare codon usage

Addressing these challenges requires systematic optimization of expression and purification protocols for this specific protein.

How can researchers troubleshoot functional assays involving L35 from I. loihiensis?

When functional assays with L35 yield inconsistent results, consider:

• Protein quality: Verify integrity using mass spectrometry and assess aggregation state with dynamic light scattering

• Buffer conditions: Systematically test different pH values, salt concentrations, and additives to identify optimal conditions

• RNA quality: Ensure RNA substrates are intact and properly folded

• Reaction specificity: Include appropriate controls (heat-denatured protein, non-specific RNA, etc.)

• Detection sensitivity: If binding signals are weak, consider alternative detection methods with higher sensitivity

• Contaminating activities: Verify the absence of nucleases or proteases that might interfere with the assay

Careful optimization and troubleshooting of assay conditions is essential for reliable functional characterization.

What considerations are important when designing experiments to study L35 function under extreme conditions?

When studying L35 under extreme conditions resembling its native environment:

• Equipment limitations: Ensure instruments can operate reliably under extreme temperature or pressure conditions

• Buffer stability: Verify that buffer components remain stable under experimental conditions

• Control proteins: Include both thermostable and non-thermostable control proteins

• Time-dependent effects: Monitor protein stability over time under extreme conditions

• Reversibility: Test whether functional changes are reversible upon return to standard conditions

• Specialized equipment: Consider using high-pressure chambers or specialized thermal cyclers designed for extremophile research

These considerations help ensure that experimental results accurately reflect the protein's native behavior under extreme conditions.

How should researchers interpret evolutionary conservation patterns in L35 across bacterial species?

When analyzing evolutionary patterns of L35:

• Distinguish between core conserved residues (likely essential for function) and variable regions (potentially involved in species-specific adaptations)

• Consider the environmental context of each species when interpreting sequence differences

• Analyze conservation in three-dimensional context, as spatial clustering of conserved residues often indicates functional importance

• Distinguish between conservation due to direct functional constraints versus structural constraints

• Use appropriate statistical models that account for the phylogenetic relationships between species

• Consider coevolution with interacting partners (rRNA, other ribosomal proteins)

This nuanced interpretation helps distinguish functionally significant variations from neutral evolutionary drift.

What statistical approaches are most appropriate for analyzing L35 binding and activity data?

For rigorous analysis of L35 functional data:

• Use appropriate binding models (Hill equation, single/multiple site models) for interpreting interaction data

• Apply Scatchard or Lineweaver-Burk plots for linearization of binding data when appropriate

• Implement global fitting approaches for complex multi-parameter models

• Consider Bayesian statistical frameworks for incorporating prior information about ribosomal proteins

• Use bootstrap or jackknife resampling to assess the robustness of parameter estimates

• Apply multiple comparison corrections (e.g., Bonferroni, false discovery rate) when comparing multiple experimental conditions

How can structural data about I. loihiensis L35 be integrated with functional and evolutionary analyses?

Integrating structural, functional, and evolutionary data requires:

• Mapping conservation scores onto three-dimensional structures to identify functionally important surfaces

• Correlating structural features with binding affinities or catalytic parameters

• Using molecular dynamics simulations to connect structure with functional dynamics

• Applying machine learning approaches to identify patterns linking sequence, structure, and function

• Creating structure-based phylogenetic trees to complement sequence-based evolutionary analyses

• Developing integrated visualizations that simultaneously display structural, functional, and evolutionary information

This integrative approach provides a more comprehensive understanding of how structure, function, and evolution are interrelated in L35.

What are promising research directions for understanding L35's role in extremophile adaptation?

Future research on I. loihiensis L35 should explore:

These directions would expand our understanding of how fundamental cellular machinery adapts to extreme conditions.

How might research on I. loihiensis L35 contribute to synthetic biology applications?

I. loihiensis L35 research could contribute to synthetic biology through:

• Engineering ribosomes with enhanced stability for industrial biotechnology applications

• Developing cell-free protein synthesis systems that function under extreme conditions

• Creating biosensors that utilize extremophile components for robust environmental detection

• Designing synthetic minimal cells capable of surviving in extreme environments

• Incorporating extremophile ribosomal components into mesophilic organisms to enhance stress tolerance

The unique adaptations of L35 from extremophiles could inspire novel biotechnological applications requiring robust cellular machinery.

What potential biotechnological applications might emerge from understanding I. loihiensis L35 structure and function?

Understanding L35 from this deep-sea extremophile could lead to:

• Development of pressure-stable enzyme systems for industrial biocatalysis

• Creation of thermotolerant protein production systems for biotechnology

• Design of stabilized therapeutic proteins with enhanced shelf-life

• Engineering of ribosomes capable of incorporating non-canonical amino acids under extreme conditions

• Advancement of astrobiology research through insights into how translation machinery might function in extreme extraterrestrial environments

These applications highlight how fundamental research on extremophile cellular components can translate into practical biotechnological innovations.