Recombinant Nitrosomonas europaea 50S ribosomal protein L15 (rplO)

What is the functional role of ribosomal protein L15 in Nitrosomonas europaea?

L15 is a 15 kDa protein of the large ribosomal subunit that plays a crucial structural and functional role in the 50S ribosomal assembly. Based on studies in other bacterial systems, L15 interacts with over ten other proteins during 50S assembly in vitro and contributes to the stabilization of ribosomal RNA tertiary structure . In N. europaea specifically, L15 likely maintains ribosomal integrity essential for protein synthesis during various metabolic states, including during the oxygen-limited conditions that this ammonia-oxidizing bacterium frequently encounters in its environmental niches .

The protein participates in the latter stages of 50S subunit assembly, helping to organize rRNA elements that are dispersed in the secondary structure. Chemical footprinting studies have shown that L15 has a strong interaction with the region spanning nucleotides 572-654 in domain II of 23S rRNA, and also interacts with specific regions of domains I, IV, and V . These multiple interaction points indicate L15's importance in maintaining the three-dimensional architecture of the ribosome.

How does the structure of L15 relate to its function in ribosome assembly?

L15 serves as a critical architectural component during ribosome biogenesis. Chemical footprinting experiments demonstrate that L15 has a strong footprint in domain II of 23S rRNA (nucleotides 572-654), but this footprint cannot be detected when L15 is incubated with "naked" 23S rRNA . This indicates that the formation of the L15 binding site requires a partially assembled particle, placing L15's incorporation at a specific point in the assembly pathway.

The three-dimensional environment of L15 within the ribosome can be characterized using protein-tethered hydroxyl radical probing. By creating L15 mutants with single cysteine residues at specific positions (such as amino acids 68, 71, and 115) and derivatizing them with 1-[p-(bromo-acetamido)benzyl]-EDTA-Fe(II), researchers can map the proximity of L15 to various rRNA elements . This approach reveals that L15 is positioned near several 23S rRNA elements that are dispersed in the secondary structure, providing important constraints on the tertiary folding of 23S rRNA and highlighting L15's role as a structural organizer.

What expression systems are most effective for producing recombinant N. europaea L15?

For successful expression of recombinant N. europaea L15, Escherichia coli-based expression systems have proven most effective when optimized appropriately. Based on similar recombinant protein expression studies, several key considerations should guide your experimental design:

Expression vector selection:

• Vectors containing T7 promoters provide controlled, high-level expression

• Including fusion tags can significantly enhance both expression and solubility

• The use of solubility-enhancing tags such as maltose-binding protein (MBP) is particularly valuable for ribosomal proteins that may have charged surfaces

Fusion tag optimization:
E. coli expression trials with various fusion proteins have shown:

The truncated maltotriose-binding protein (MBP) from Pyrococcus furiosus (residues 59-433) has shown particular promise as a solubility-enhancing tag when combined with a modified histidine tag (HE) . For optimal results, include a tobacco etch virus (TEV) protease recognition site between the tag and L15 to facilitate tag removal.

What are the optimal conditions for purifying recombinant L15 while maintaining its native structure?

Purification of recombinant L15 requires careful consideration of buffer conditions and purification strategies to maintain the protein's native structure. A multi-step purification approach is recommended:

Initial capture by immobilized metal affinity chromatography (IMAC):

• For His-tagged constructs, use Ni²⁺-charged resins with imidazole gradients (20-250 mM)

• For CusF-tagged constructs, Cu²⁺-charged resins have shown excellent results

• Include reducing agents (1-5 mM DTT or 2-10 mM β-mercaptoethanol) to prevent oxidation

Buffer optimization:
Since L15 naturally interacts with RNA, consider adding the following stabilizers:

• Moderate ionic strength (150-300 mM NaCl) to minimize non-specific interactions

• Glycerol (5-10%) to enhance stability during concentration steps

• Low concentrations of Mg²⁺ (1-5 mM) to mimic the ribosomal environment

Tag removal considerations:

• The dipeptidyl aminopeptidase 1 (DAP-1) system has shown complete removal of specific tags within 4-12 hours under mild conditions

• For TEV protease cleavage, optimize conditions (enzyme:substrate ratio, temperature, time) based on fusion construct design

• After cleavage, perform reverse IMAC to separate the cleaved protein from the tag and uncut fusion protein

Final polishing:

• Size exclusion chromatography to ensure homogeneity and remove aggregates

• Quality assessment by SDS-PAGE, mass spectrometry, and dynamic light scattering

When designing your purification strategy, remember that L15's natural binding partners include rRNA and other ribosomal proteins. Therefore, RNase treatment during lysis may be necessary to prevent co-purification of E. coli RNA, which could affect downstream applications.

How can I verify that purified recombinant L15 maintains its native conformation and activity?

Verifying the native conformation and activity of purified recombinant L15 requires multiple complementary approaches:

Structural integrity assessment:

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

• Thermal shift assays to determine stability and proper folding

• Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to confirm the monomeric state and detect any oligomerization

Functional assessment through RNA binding studies:

• Electrophoretic mobility shift assays (EMSA) with the 23S rRNA domain II fragment (nucleotides 572-654)

• Chemical footprinting using Fe(II)·EDTA and dimethyl sulfate to verify protection of expected 23S rRNA regions

• Isothermal titration calorimetry (ITC) to measure binding affinity and thermodynamic parameters

In vitro reconstitution activity:
L15's primary function is in ribosome assembly, so the gold standard for functional assessment is its ability to incorporate into 50S subunits. This can be tested by:

• Reconstituting core particles derived from N. europaea or E. coli 50S subunits (by treatment with 2M LiCl)

• Analyzing reconstituted particles by sucrose gradient ultracentrifugation

• Confirming L15 incorporation by western blotting and mass spectrometry analysis

A critical observation from previous research is that L15's binding site cannot be detected when incubated with "naked" 23S rRNA, indicating that formation of the L15 binding site requires a partially assembled particle . Therefore, binding assays should ideally use partially reconstituted core particles rather than isolated rRNA.

What techniques can map the interaction between L15 and 23S rRNA in N. europaea ribosomes?

Mapping the precise interactions between L15 and 23S rRNA requires specialized techniques that can provide atomic or near-atomic resolution data. Based on established methodologies, the following approaches are most effective:

Chemical footprinting:
This approach identifies RNA regions protected by protein binding:

• Reconstitute purified recombinant L15 with core particles derived from N. europaea 50S subunits

• Expose the complexes to chemical probes such as Fe(II)·EDTA and dimethyl sulfate

• Analyze the modification patterns by primer extension or next-generation sequencing

• Compare patterns with and without L15 to identify protected regions

Previous studies using this approach identified a strong footprint for L15 in the region spanning nucleotides 572-654 in domain II of 23S rRNA .

Protein-tethered hydroxyl radical probing:
This technique provides detailed information about the three-dimensional environment of L15:

• Create L15 mutants with single cysteine residues at specific positions (e.g., amino acids 68, 71, and 115)

• Derivatize these mutants with 1-[p-(bromo-acetamido)benzyl]-EDTA·Fe(II)

• Reconstitute with core particles and initiate hydroxyl radical cleavage

• Map the RNA cleavage sites by primer extension or sequencing

This approach has revealed cleavages not only in domain II but also in specific regions of domains I, IV, and V of 23S rRNA, indicating the proximity of these rRNA regions to one another and providing constraints on the tertiary folding of 23S rRNA .

Cryo-electron microscopy (cryo-EM):
For a comprehensive structural view, cryo-EM provides near-atomic resolution of the entire ribosomal complex:

• Reconstitute 50S subunits with recombinant L15

• Prepare vitrified samples for cryo-EM analysis

• Collect and process images to generate 3D reconstructions

• Build atomic models that reveal the positioning of L15 within the ribosomal context

How can site-directed mutagenesis of L15 be used to investigate its role in ribosome assembly?

Site-directed mutagenesis offers a powerful approach to probe the functional importance of specific residues in L15. A systematic mutagenesis strategy should target residues in key regions:

Experimental design strategy:

• Identify critical residues based on:

  • Conserved amino acids identified through sequence alignment with L15 homologs

  • Residues positioned near cysteine mutations used in hydroxyl radical probing (positions 68, 71, and 115)

  • Charged or aromatic residues that might mediate RNA interactions

• Create single-point mutations using PCR-based mutagenesis approaches:

  • Conservative substitutions (e.g., Lys→Arg) to test charge importance

  • Non-conservative substitutions (e.g., Lys→Ala) to eliminate side chain functionality

  • Introduce cysteine residues at positions of interest for subsequent hydroxyl radical probing

• Assess mutant effects through multiple functional assays:

• Interpret results in structural context:

  • Map mutations onto 3D models derived from cryo-EM or homology modeling

  • Correlate functional defects with structural perturbations

  • Identify networks of interactions that contribute to ribosome assembly

This approach can reveal which aspects of L15 structure are essential for its role in ribosome assembly and which might be adaptable, providing insights into both universal features of ribosome assembly and any specific adaptations in N. europaea.

How does L15 from N. europaea compare to its homologs in other bacterial species?

Comparing L15 from N. europaea with homologs from other bacteria provides insights into both conserved functional elements and species-specific adaptations. A comprehensive comparison should include:

Sequence conservation analysis:
While the search results don't provide specific sequence data for N. europaea L15, ribosomal proteins are generally highly conserved due to their fundamental roles. Key comparisons should include:

• Alignment with model organisms (E. coli, B. subtilis) to identify core conserved regions

• Comparison with other ammonia-oxidizing bacteria to identify niche-specific adaptations

• Identification of N. europaea-specific insertions or deletions that might influence function

Structural comparison:
Using available structural data or homology models:

• Map conserved residues onto 3D structures to identify functionally critical regions

• Analyze surface properties (electrostatic potential, hydrophobicity) that might influence rRNA binding

• Examine any structural features unique to N. europaea that might relate to its ecological niche

Functional conservation:
Experimental approaches to compare functional properties:

• Express recombinant L15 proteins from different species and compare biochemical properties

• Test cross-species complementation in reconstitution assays

• Measure binding affinities to conserved rRNA regions using ITC or SPR

These comparative analyses can reveal how L15 has evolved in N. europaea to support its specialized metabolism as an ammonia-oxidizing bacterium, potentially including adaptations related to oxygen limitation or other environmental factors relevant to its ecological niche .

How can recombinant L15 be used to study the effects of environmental stressors on N. europaea ribosome function?

N. europaea encounters various environmental stressors in its natural habitats, including oxygen limitation, which significantly affects its metabolism . Recombinant L15 provides a valuable tool for investigating how these stressors impact ribosome function:

Experimental approaches:

• Stress-specific modification analysis:

  • Purify ribosomes from N. europaea grown under different stress conditions (oxygen limitation, ammonia starvation, pH stress)

  • Extract and analyze L15 by mass spectrometry to identify stress-induced post-translational modifications

  • Create recombinant L15 variants mimicking these modifications to test their functional impact

• Ribosome heterogeneity assessment:

  • Use recombinant tagged L15 to purify intact ribosomes under different conditions

  • Analyze ribosome composition to detect stress-induced changes in ribosomal protein stoichiometry

  • Investigate whether environmental stressors lead to "specialized ribosomes" with altered composition

• Translation fidelity and efficiency studies:

  • Reconstitute ribosomes with stress-modified L15 or L15 variants

  • Measure translation rates and error frequencies using reporter systems

  • Determine if L15 modifications contribute to selective translation of stress-response proteins

• Integration with transcriptomic data:
  Correlate ribosomal adaptations with broader cellular responses:

These studies can reveal how N. europaea modulates its translational machinery through L15 modifications to adapt to environmental challenges, with implications for understanding this organism's ecological role in nitrification processes.

How does L15 contribute to N. europaea's adaptation to oxygen-limited conditions?

N. europaea experiences frequent oxygen limitation in its environmental niches, which significantly affects its metabolism and transcriptome . L15 may play important roles in adaptation to these conditions:

Transcriptomic context:
Under oxygen-limited conditions, N. europaea exhibits various metabolic adaptations:

• Reduced growth yield and non-stoichiometric ammonia-to-nitrite conversion

• Upregulation of both heme-copper-containing cytochrome c oxidases

• Significant increase in transcription of B-type heme-copper oxidase (proposed to function as a nitric oxide reductase)

L15's potential role in metabolic adaptation:

• Translational regulation:

  • L15 modifications under oxygen limitation may affect ribosome function

  • Changes in L15 incorporation could alter translation of specific mRNAs related to energy metabolism

  • Hypothesize that L15 contributes to selective translation of oxidases upregulated during oxygen limitation

• Energy conservation connection:
  Transcriptomic data shows differential transcription of polyphosphate metabolism-related genes under oxygen limitation, with increased synthesis and unchanged degradation . This suggests:

  • ATP consumption pathways may be modulated through translational control

  • L15 modifications might influence translation efficiency of energy-related genes

  • Ribosome function may be optimized for energy conservation under stress

• Experimental approaches:

  • Create reporter systems to monitor translation of specific mRNAs under oxygen limitation

  • Use ribosome profiling to map translation events across the transcriptome under different oxygen conditions

  • Isolate ribosomes from oxygen-limited cultures and characterize L15 status (modifications, incorporation)

Understanding L15's contribution to oxygen limitation response would provide insights into the molecular mechanisms underlying N. europaea's ecological versatility in environments with fluctuating oxygen levels.

How can recombinant L15 be used to develop biosensors for monitoring N. europaea activity in environmental samples?

Recombinant L15 protein can serve as a foundation for developing biosensors to monitor N. europaea activity in complex environmental samples. The approach draws inspiration from bioluminescence assays developed for N. europaea but focuses specifically on L15-based detection systems:

Design principles for L15-based biosensors:

• Reporter fusion approaches:

  • Create C-terminal fusions of L15 with reporter proteins (GFP, luciferase)

  • Design expression vectors where the L15 promoter drives reporter gene expression

  • Develop split reporter systems where complementary fragments are fused to L15 and interacting partners

• Detection strategies:

  Biosensor Type	Detection Method	Sensitivity	Applications
  L15-luciferase fusion	Bioluminescence	High	Real-time monitoring
  L15 promoter-GFP	Fluorescence	Medium	Population dynamics
  L15-aptamer systems	Electrochemical	Very high	Field detection

• Validation approaches:

  • Calibrate biosensor response against established methods for measuring N. europaea activity

  • Test specificity by comparing responses between N. europaea and related/unrelated bacteria

  • Validate in complex environmental matrices (soil, wastewater, aquatic samples)

The bioluminescence assay approach described for N. europaea provides a useful model, as it successfully expressed the Vibrio harveyi luxAB genes in N. europaea, resulting in observable light emission that responded to inhibitors . A similar approach using L15 as the basis for detection could provide specific information about ribosome status and protein synthesis activity in environmental samples.

What experimental approaches can assess how L15 contributes to nitrification efficiency in N. europaea?

L15's role in maintaining ribosomal function may significantly impact nitrification efficiency in N. europaea. Exploring this connection requires integrative experimental approaches:

Genetic manipulation strategies:

• Create conditional knockdown strains of L15 in N. europaea (if genetic tools available)

• Develop strains expressing modified versions of L15 with altered functionality

• Use CRISPR interference (CRISPRi) to modulate L15 expression levels

Physiological assessment:
Measure key parameters of nitrification efficiency:

• Ammonia oxidation rates under different L15 expression levels

• Nitrite production kinetics and yield coefficients

• Growth yield and coupling between ammonia oxidation and growth

• Production of intermediates (hydroxylamine) and byproducts (NO, N₂O)

Mechanistic investigations:
Connect L15 function to nitrification biochemistry:

• Analyze expression levels and translation efficiency of key nitrification enzymes:

  • Ammonia monooxygenase (AMO)

  • Hydroxylamine oxidoreductase (HAO)

  • Nitrite reductase (NirK)

• Monitor enzyme activities in relation to L15 status:

  • Measure specific activities of purified enzymes

  • Assess in vivo electron flow through the respiratory chain

  • Quantify the impact on ATP generation and energy conservation

• Integrate with transcriptomic data:
  Previous studies have shown that under oxygen-limited conditions, N. europaea exhibits reduced growth yield and non-stoichiometric ammonia-to-nitrite conversion, alongside changes in gene expression patterns . Investigating how L15 influences translation of these differentially expressed genes would provide insights into ribosome-mediated adaptation mechanisms.

These approaches would reveal how L15-dependent ribosomal function contributes to N. europaea's ecological role in nitrification and nitrogen cycling.

How does L15 expression change during different growth phases and environmental conditions in N. europaea?

Understanding how L15 expression responds to growth phases and environmental conditions provides insights into ribosomal adaptation in N. europaea. A comprehensive analysis would include:

Expression profiling across growth conditions:

• Growth phase effects:

  • Monitor L15 mRNA and protein levels throughout batch culture (lag, exponential, stationary phases)

  • Compare with other ribosomal proteins to identify differential regulation

  • Correlate with ribosome abundance and activity

• Environmental condition responses:

  Condition	L15 Expression	Other Ribosomal Proteins	Correlation with Metabolism
  Oxygen limitation	?	?	Changes in cytochrome oxidase expression
  Ammonia limitation	?	?	Changes in ammonia transport/utilization
  pH stress	?	?	Changes in membrane integrity genes
  Temperature shifts	?	?	Changes in chaperone expression

• Specific responses during oxygen limitation:
  Based on transcriptomic data, N. europaea shows significant metabolic adjustments under oxygen limitation . Investigating whether L15 is differentially regulated compared to other ribosomal proteins could reveal specialized roles in adaptation.

Methodological approaches:

• Gene expression analysis:

  • RT-qPCR targeting L15 mRNA under different conditions

  • RNA-seq for transcriptome-wide context

  • Ribosome profiling to assess translation efficiency

• Protein level analysis:

  • Western blotting with L15-specific antibodies

  • Quantitative proteomics using stable isotope labeling

  • Analysis of post-translational modifications by mass spectrometry

• In situ visualization:

  • Fluorescent protein fusions to monitor L15 localization and abundance

  • Single-molecule FISH to detect mRNA distribution

  • Correlative light and electron microscopy to connect expression with ultrastructure

This multi-level analysis would reveal whether L15 exhibits condition-specific regulation that contributes to N. europaea's environmental adaptability, particularly in the context of its role as an ammonia-oxidizing bacterium in the nitrogen cycle.

What strategies can overcome the challenges of expressing and purifying highly basic ribosomal proteins like L15?

Ribosomal proteins including L15 are often highly basic, which creates specific challenges for recombinant expression and purification. Based on successful approaches for similar proteins, the following strategies are recommended:

Expression optimization:

• Codon optimization:

  • Adjust codon usage for N. europaea L15 to match E. coli preferences

  • Avoid rare codons, particularly for arginine and lysine, which are abundant in basic proteins

• Fusion partner selection:

  • Utilize acidic fusion partners to balance the highly basic nature of L15

  • The His-MBP fusion approach has proven effective for basic proteins

  • Consider the HE-MBP(Pyr) system, which has shown high levels of soluble expression for challenging proteins

• Expression conditions:

  • Lower induction temperatures (15-18°C) to slow synthesis and improve folding

  • Use auto-induction media for gradual protein expression

  • Co-express with chaperones to assist proper folding

Purification challenges and solutions:

• RNA contamination:

  • Basic proteins like L15 often co-purify with host E. coli RNA

  • Implement high-salt washes (0.5-1M NaCl) during initial purification steps

  • Include RNase treatment during lysis and early purification steps

  • Consider benzonase nuclease treatment to eliminate all nucleic acids

• Charge-based separation:

  • Utilize cation exchange chromatography after initial IMAC purification

  • Apply heparin affinity chromatography, which works well for nucleic acid-binding proteins

  • Develop a polyethyleneimine (PEI) precipitation step to remove nucleic acids before chromatography

• Stability enhancement:

  • Maintain reducing conditions throughout purification (5mM DTT or 10mM β-mercaptoethanol)

  • Include stabilizing agents like arginine (50-100mM) to prevent aggregation

  • Use buffers that mimic the ribosomal environment (moderate salt, Mg²⁺, polyamines)

These strategies address the specific challenges associated with basic ribosomal proteins and can significantly improve the yield and purity of recombinant L15.

How can I optimize reconstitution of L15 into ribosomal particles for structural and functional studies?

Reconstituting L15 into ribosomal particles is essential for structural and functional studies but presents several technical challenges. Based on approaches described in the literature, the following optimization strategies are recommended:

Preparation of ribosomal components:

• Core particle preparation:

  • Isolate 50S subunits from N. europaea or E. coli (if using as a surrogate system)

  • Generate core particles by treatment with 2M LiCl, which removes a subset of proteins including L15

  • Verify that core particles maintain a compact 50S-like structure by sucrose gradient analysis

• rRNA preparation alternatives:

  • If using isolated rRNA, ensure proper refolding by controlled cooling from denaturing conditions

  • Include magnesium and monovalent ions at physiological concentrations

  • Consider preparing rRNA fragments corresponding to L15 binding domains for initial binding studies

Reconstitution protocol optimization:

• Sequential assembly approach:

  • Recognize that L15 binding requires a partially assembled particle

  • Establish the correct order of protein addition based on assembly maps

  • Include any assembly cofactors identified in the literature

• Buffer optimization:

  Component	Concentration Range	Function
  Mg²⁺	10-20 mM	Stabilizes rRNA tertiary structure
  NH₄⁺/K⁺	100-200 mM	Promotes proper folding
  Spermidine	2-5 mM	Stabilizes RNA-protein interactions
  HEPES/Tris	20-50 mM (pH 7.5-8.0)	Maintains optimal pH

• Temperature and time considerations:

  • Perform initial binding at low temperature (0-4°C) followed by controlled warming

  • Allow sufficient incubation time (30-60 minutes) for proper incorporation

  • Consider thermal cycling protocols that promote correct assembly

Verification of reconstitution:

• Functional verification:

  • Apply chemical footprinting using Fe(II)·EDTA and dimethyl sulfate to confirm L15 incorporation

  • Assess protection of expected rRNA regions (nucleotides 572-654 in domain II)

  • Compare footprinting patterns with those of native 50S subunits

• Structural verification:

  • Analyze reconstituted particles by electron microscopy (negative staining or cryo-EM)

  • Perform sucrose gradient ultracentrifugation to confirm the sedimentation properties

  • Use protein-specific antibodies to verify L15 incorporation by Western blotting

These approaches address the specific challenges of reconstituting L15 into ribosomal particles and provide multiple methods to verify successful reconstitution.

What analytical techniques can verify the structural integrity of recombinant L15 compared to the native protein?

Verifying that recombinant L15 maintains the same structural integrity as the native protein is critical for ensuring the validity of experimental findings. A comprehensive analytical approach includes:

Primary structure verification:

• Mass spectrometry analysis:

  • Intact mass analysis to confirm the expected molecular weight

  • Peptide mapping by tryptic digestion and LC-MS/MS

  • Identification of any post-translational modifications present in native L15

• N-terminal sequencing:

  • Verify correct processing of fusion tags after cleavage

  • Confirm the absence of unexpected modifications or truncations

Secondary and tertiary structure analysis:

• Spectroscopic techniques:

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

  • Fluorescence spectroscopy to assess tertiary structure (if L15 contains tryptophan residues)

  • FTIR spectroscopy for complementary secondary structure information

• Stability assessment:

  • Thermal shift assays (Thermofluor) to compare melting temperatures

  • Chemical denaturation curves using intrinsic fluorescence

  • Limited proteolysis patterns to identify domain boundaries and stability

Functional equivalence testing:

• RNA binding assays:

  • Electrophoretic mobility shift assays with 23S rRNA fragments

  • Chemical footprinting to compare protection patterns between recombinant and native L15

  • Isothermal titration calorimetry to measure binding thermodynamics

• In vitro reconstitution:

  • Compare the ability of recombinant and native L15 to incorporate into core particles

  • Assess reconstituted particle structure by electron microscopy

  • Functional testing of reconstituted ribosomes in translation assays

These analytical techniques provide a comprehensive assessment of structural integrity across multiple levels of protein structure, ensuring that recombinant L15 accurately represents the native protein for experimental studies.