Recombinant Protochlamydia amoebophila 50S ribosomal protein L19 (rplS)

What is the primary structure and basic characteristics of Protochlamydia amoebophila rplS?

Protochlamydia amoebophila UWE25 rplS is a 135-amino acid protein with a molecular weight of approximately 15.2 kDa. Its primary sequence is:
MIMSRSAIIEKLQNEQMKKDITPFRIGDTVRVHIRIIEGDKERTQVFAGTVIARKGQGLSETFSVHRVAYGEGMERVFMLHSPRIAKIEVIKEGDVRRSKLYYLRGTSGKASKVKARLGARRSSNAVTKQIVSAE

This protein belongs to the bacterial ribosomal protein bL19 family and serves as a structural component of the 50S ribosomal subunit. Unlike its E. coli counterpart which contains 114 amino acids , P. amoebophila rplS has 135 amino acids, suggesting potential structural or functional adaptations specific to this organism.

What expression systems are most effective for recombinant production of P. amoebophila rplS?

Based on available research methodologies for similar proteins, the following approach is recommended for recombinant expression of P. amoebophila rplS:

• Expression vector selection: The pET16b vector system has been successfully used for expression of other P. amoebophila proteins, adding an N-terminal His tag to facilitate purification . This system would likely be effective for rplS expression as well.

• Host selection: E. coli BL21(DE3) has proven effective for expression of P. amoebophila proteins . This strain is particularly suitable because it lacks certain proteases that might degrade the recombinant protein.

• Expression conditions: Optimal expression is typically achieved at room temperature following induction with 1 mM IPTG . This moderate temperature helps prevent inclusion body formation while maintaining good protein yield.

• Purification strategy: HisTrap purification columns (such as HiTrap HP) provide efficient purification of His-tagged recombinant proteins . For rplS specifically, prepare to:

  • Lyse cells in appropriate buffer with protease inhibitors

  • Perform affinity chromatography using nickel columns

  • Verify purified protein by SDS-PAGE and mass spectrometry

When designing constructs, consider excluding hydrophobic domains if present, as was done with other P. amoebophila proteins to improve solubility and expression efficiency .

What are the methodological considerations for structural studies of rplS?

When investigating the structural properties of P. amoebophila rplS, researchers should consider:

• Secondary structure prediction: Tools such as the Kyte-Doolittle hydrophilicity profile (window size of seven) through the MacVector software can be utilized to analyze hydrophobicity patterns . For coiled-coil structures, MARCOIL prediction tools would be appropriate .

• Crystallization approaches: Due to its relatively small size (15.2 kDa), rplS may be amenable to X-ray crystallography following high-purity preparation. Consider:

  • Screening various crystallization conditions (pH, salt concentration, temperature)

  • Co-crystallization with RNA fragments or interacting partners

  • Surface entropy reduction mutations if crystallization proves difficult

• Structure-function correlation: To understand functional domains, combine structural data with:

  • Site-directed mutagenesis of conserved residues

  • Ribosome binding assays to measure functional impacts

  • Comparative analysis with homologous proteins from other bacteria

How does Protochlamydia amoebophila relate to other Chlamydiae, and what is the evolutionary significance of its ribosomal proteins?

Protochlamydia amoebophila UWE25 represents an important evolutionary link within the Chlamydiae phylum. Unlike the pathogenic Chlamydiaceae that primarily infect mammalian and avian hosts, P. amoebophila functions as an endosymbiont of free-living amoebae . This ecological distinction has significant implications:

• Evolutionary position: P. amoebophila represents a more ancient lineage of Chlamydiae, providing insights into the evolutionary history of this bacterial group before the adaptation to animal hosts.

• Gene contribution to eukaryotes: Phylogenomic analyses have identified at least 55 Chlamydiae-derived genes in algae and plants, suggesting significant genetic contributions from ancient Chlamydiae to Plantae . This indicates a potentially important role of Chlamydiae in eukaryotic evolution.

• Ribosomal diversity context: Within Chlamydiae, there can be significant diversity in ribosomal RNA genes. For example, "Candidatus Protochlamydia amoebophila" contains three 16S rRNA genes with notable diversity at the secondary structural level . This diversity context is important when studying specific ribosomal proteins like rplS.

The evolutionary conservation of ribosomal proteins like rplS can provide insights into both the core translational machinery maintained across bacterial lineages and the specific adaptations that occurred during the evolution of obligate intracellular lifestyles.

What can comparative genomics reveal about rplS conservation across bacterial species?

Comparative genomic approaches to studying rplS conservation should include:

• Sequence alignment methodology:

  • Collect rplS homologs from diverse bacterial phyla

  • Perform multiple sequence alignment using MUSCLE or CLUSTAL

  • Identify conserved domains and variable regions

  • Calculate amino acid identity values through pairwise alignment methods

• Phylogenetic analysis approach:

  • Construct maximum likelihood trees using RAxML with the WAG substitution model

  • Implement the PROTGAMMA (+Γ) algorithm with 4 discrete rate categories

  • Perform bootstrap analysis (100-200 replicates) to assess node reliability

  • Use tools like PhyloSort to identify specific topological relationships

• Structural conservation analysis:

  • Map sequence conservation onto predicted structures

  • Identify structurally constrained regions versus variable regions

  • Correlate with known functional domains in ribosomal proteins

This approach would reveal whether rplS shows evidence of horizontal gene transfer, which has been documented for other genes in the evolutionary history of Chlamydiae .

How might rplS research contribute to understanding P. amoebophila's intracellular lifestyle?

Investigating rplS in P. amoebophila could provide insights into several aspects of this organism's obligate intracellular lifestyle:

• Specialized translation machinery: As an obligate intracellular bacterium, P. amoebophila may exhibit adaptations in its translation apparatus, including ribosomal proteins like rplS. Comparative analysis with free-living bacteria could reveal specialized features.

• Host interaction context: While rplS itself is not an inclusion membrane protein, P. amoebophila does possess inclusion membrane proteins (designated IncA, IncQ, IncR, and IncS) that modify the membrane of its host-derived vesicular compartment . Understanding how core cellular functions like translation operate in this specialized intracellular environment is crucial.

• Experimental approaches:

  • Immunofluorescence analysis to determine rplS localization during infection

  • Co-immunoprecipitation to identify interaction partners unique to intracellular growth

  • Transcriptional analysis to examine expression patterns during different stages of the infection cycle

What methodological approaches can detect potential moonlighting functions of rplS beyond translation?

Several ribosomal proteins have been found to perform secondary functions beyond their role in translation. To investigate such possibilities for P. amoebophila rplS:

• Protein-protein interaction studies:

  • Yeast two-hybrid screening using rplS as bait against P. amoebophila and host cell libraries

  • Pull-down assays with tagged recombinant rplS followed by mass spectrometry

  • In situ proximity labeling (BioID or APEX) to identify neighbors in the cellular context

• Subcellular localization:

  • Generate antibodies against purified recombinant rplS for immunofluorescence analysis

  • Perform cell fractionation studies to detect potential non-ribosomal pools of rplS

  • Use immuno-transmission electron microscopy for precise localization

• Functional assays:

  • RNA binding studies to identify potential regulatory roles

  • DNA binding assays to test for possible transcription factor activity

  • Stress response analysis to detect condition-specific relocalization

What are the most effective purification strategies for native rplS from P. amoebophila?

Purifying native rplS directly from P. amoebophila presents significant challenges due to its obligate intracellular lifestyle. An effective methodological approach would include:

• Cell culture and harvest:

  • Cultivate P. amoebophila in amoeba hosts (e.g., Acanthamoeba sp.)

  • Harvest bacteria at optimal growth phase

  • Purify bacteria from host cells through density gradient centrifugation

• Ribosome isolation:

  • Lyse bacteria under RNase-free conditions

  • Separate crude ribosomes through ultracentrifugation

  • Purify 50S subunits using sucrose gradient separation

• Protein extraction and identification:

  • Extract ribosomal proteins using acetic acid or lithium chloride

  • Separate proteins using reversed-phase HPLC

  • Confirm identity by mass spectrometry and Western blotting with specific antibodies

This approach would yield native rplS in its physiological context, although with lower yield than recombinant expression systems.

How can researchers effectively design experiments to study rplS interactions with other ribosomal components?

To investigate the interactions between rplS and other ribosomal components, consider these methodological approaches:

• In vitro reconstitution assays:

  • Express and purify recombinant rplS and potential interaction partners

  • Perform binding assays under various conditions (pH, salt, temperature)

  • Use techniques like surface plasmon resonance or isothermal titration calorimetry to determine binding kinetics and thermodynamics

• Cross-linking studies:

  • Apply RNA-protein crosslinking methods to capture interactions with rRNA

  • Use protein-protein crosslinkers of various lengths to identify neighboring proteins

  • Analyze crosslinked complexes by mass spectrometry

• Mutational analysis:

  • Create point mutations in conserved residues of rplS

  • Assess effects on ribosome assembly and function

  • Perform complementation studies in suitable model systems

• Structural biology approaches:

  • Use cryo-electron microscopy to visualize rplS in the context of the whole ribosome

  • Employ NMR to study dynamic interactions in solution

  • Apply hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

What statistical approaches are most appropriate for analyzing rplS sequence conservation data?

When analyzing sequence conservation patterns of rplS across bacterial species, researchers should employ the following statistical approaches:

• Sequence conservation metrics:

  • Calculate per-site entropy scores to quantify variability

  • Use conservation indices (e.g., Valdar's weighted sum-of-pairs measure)

  • Apply rate4site algorithm to estimate evolutionary rates at each position

• Comparative statistical methods:

  • Implement maximum likelihood models of sequence evolution

  • Calculate dN/dS ratios to detect selective pressure

  • Use Bayesian approaches to estimate posterior probabilities of conservation

• Visualization and interpretation:

  • Generate sequence logos to represent conservation patterns

  • Map conservation scores onto structural models

  • Cluster sequences based on similarity patterns using appropriate distance metrics

These approaches will help distinguish between functionally constrained regions and those under relaxed selection, providing insights into the protein's evolution.

How should researchers design experiments to investigate potential post-translational modifications of rplS?

Post-translational modifications (PTMs) can significantly affect protein function. To investigate PTMs in P. amoebophila rplS:

• Mass spectrometry-based approach:

  • Purify native or recombinant rplS under conditions that preserve PTMs

  • Perform tryptic digestion followed by LC-MS/MS analysis

  • Use multiple fragmentation methods (CID, ETD, HCD) for comprehensive coverage

  • Employ targeted methods like multiple reaction monitoring for specific modifications

• PTM-specific detection methods:

  • Use phospho-specific staining or antibodies for phosphorylation

  • Apply glycan-specific detection reagents for glycosylation

  • Implement acetylation-specific antibodies for acetylation sites

• Functional validation:

  • Create site-directed mutants that mimic or prevent modification

  • Assess functional consequences through in vitro translation assays

  • Compare modification patterns under different growth conditions

• Data analysis considerations:

  • Use appropriate search algorithms with variable modification parameters

  • Apply false discovery rate controls for modification site assignment

  • Implement quantitative approaches to determine stoichiometry of modifications