Recombinant Nocardia farcinica 50S ribosomal protein L3 (rplC)

What is the structure and function of Nocardia farcinica 50S ribosomal protein L3 (rplC)?

The 50S ribosomal protein L3 (rplC) from Nocardia farcinica is a primary rRNA binding protein that directly binds near the 3'-end of the 23S rRNA, where it nucleates assembly of the 50S ribosomal subunit. The protein consists of 221 amino acids with a molecular mass of approximately 23.2 kDa . The complete amino acid sequence is: MTDNKNRPAAGILGTKLGMTQVFDEKNRVVPVTVIKAGPNVVTQIRTEERDGYSAVQVAFGAIDPRKVNKPVAGQFAKAGVTPRRHIAEIRVADASSFEVGQEINADVFEEGSYVDVTGTSKGKGYAGTMKRHGFRGQGASHGAQAVHRRPGSIGGCATPGRVFKGMRMAGRMGNDRVTTQNLSVHKVDAENGLLLIKGAIPGRKGGVVIVKSAVKGGAHA . It belongs to the universal ribosomal protein uL3 family, which is highly conserved across species due to its essential role in ribosome assembly and function. As a primary binding protein, rplC serves as a nucleation point for the assembly of other ribosomal components, making it crucial for proper ribosome structure and protein synthesis.

How does rplC contribute to the antibiotic resistance mechanisms in Nocardia farcinica?

While rplC itself is not directly identified in the provided search results as an antibiotic resistance determinant in Nocardia farcinica, the bacterium demonstrates resistance to several antibiotics through various mechanisms. N. farcinica shows natural resistance to multiple antibiotics including β-lactams, aminoglycosides, macrolides, and rifamycins . One specific resistance mechanism involves the rox gene, which encodes a rifampicin monooxygenase capable of converting rifampicin to 2'-N-hydroxy-4-oxo-rifampicin with markedly lowered antibiotic activity .

Ribosomal proteins like rplC can potentially contribute to antibiotic resistance by altering the binding sites of antibiotics that target the ribosome. Mutations in ribosomal proteins or rRNA can modify the structure of the ribosome, potentially reducing the affinity of antibiotics. For comprehensive research into potential roles of rplC in antibiotic resistance, researchers would need to conduct targeted mutation studies or structural analyses of the protein in the presence of various antibiotics.

What expression systems are most effective for producing recombinant N. farcinica rplC?

Based on the search results, E. coli is utilized as an expression system for recombinant N. farcinica rplC protein production . E. coli represents an effective expression system due to its rapid growth, well-characterized genetics, and established protocols for protein expression. When expressing rplC, researchers should consider the following methodological approaches:

• Codon optimization: Adjust the coding sequence to match E. coli codon usage preferences for improved expression.

• Expression vector selection: Use vectors with strong, inducible promoters (e.g., T7) for controlled expression.

• Fusion tags: Consider expressing the protein with affinity tags (His-tag, GST, etc.) to facilitate purification.

• Growth conditions: Optimize temperature, induction time, and inducer concentration to maximize protein yield while maintaining proper folding.

• Purification strategy: Implement a multi-step purification process, potentially including affinity chromatography and size exclusion chromatography.

For long-term storage of the purified protein, adding glycerol to a final concentration of 5-50% and storing at -20°C/-80°C is recommended. According to the product information, the shelf life of the liquid form is typically 6 months at -20°C/-80°C, while the lyophilized form can remain stable for up to 12 months .

How can researchers utilize rplC in studies of bacterial ribosomal assembly and protein synthesis inhibition?

Researchers can leverage recombinant N. farcinica rplC for multiple advanced applications in studying ribosomal assembly and protein synthesis inhibition:

In vitro ribosome reconstitution studies:

• Using purified rplC along with other ribosomal proteins and rRNA to reconstitute functional ribosomal subunits.

• Monitoring the assembly process using techniques such as light scattering, sedimentation analysis, or cryo-electron microscopy.

• Introducing mutations in rplC to identify critical residues for ribosome assembly and function.

Antibiotic binding and resistance mechanisms:

• Conducting binding assays with labeled antibiotics that target the ribosome to determine if rplC is involved in antibiotic binding.

• Creating site-directed mutants of rplC to identify residues important for antibiotic sensitivity or resistance.

• Performing structural studies (X-ray crystallography or cryo-EM) of rplC in complex with antibiotics to elucidate binding interfaces.

Protein-RNA interaction studies:

• Using techniques such as RNA footprinting, electrophoretic mobility shift assays, or surface plasmon resonance to characterize the interaction between rplC and 23S rRNA.

• Mapping the binding interface through chemical cross-linking followed by mass spectrometry.

• Investigating how rplC nucleates the assembly of the 50S subunit through temporal assembly studies.

These approaches provide valuable insights into fundamental ribosomal biology and potential targets for new antimicrobial agents.

What are the implications of rplC mutations for antibiotic development against Nocardia infections?

Mutations in ribosomal proteins like rplC can significantly impact antibiotic efficacy and represent important considerations for drug development. For researchers investigating new antibiotics against Nocardia infections, several methodological approaches should be considered:

Mutation analysis methodology:

• Generate site-directed mutations in conserved regions of rplC that might interact with antibiotics.

• Express mutant proteins in susceptible bacterial strains to assess changes in antibiotic susceptibility.

• Perform minimum inhibitory concentration (MIC) assays comparing wild-type and mutant strains.

• Use structural modeling to predict how mutations might alter antibiotic binding sites.

Clinical implications:
N. farcinica is clinically significant as it causes localized and disseminated infections, particularly in immunocompromised patients . It demonstrates resistance to several extended-spectrum antimicrobial agents, making identification and targeted treatment crucial . Understanding rplC's role in ribosome function could reveal new antibiotic targets or help understand existing resistance mechanisms.

Research applications:

• Screen chemical libraries for compounds that specifically target unique features of N. farcinica rplC.

• Develop combination therapies that target multiple components of the bacterial ribosome.

• Design peptidomimetic inhibitors that disrupt rplC's interaction with other ribosomal components.

These approaches may ultimately lead to novel therapeutic strategies for combating Nocardia infections, which are particularly important for immunocompromised patients where treatment options may be limited.

How does the structure-function relationship of rplC compare between Nocardia farcinica and other bacterial species?

A comparative analysis of rplC structure and function across bacterial species reveals important evolutionary and functional insights:

Sequence conservation analysis:
The universal ribosomal protein uL3 family, to which N. farcinica rplC belongs, is highly conserved across bacterial species . Researchers should conduct:

• Multiple sequence alignments to identify conserved domains and species-specific variations.

• Phylogenetic analysis to understand evolutionary relationships.

• Structural prediction to map conserved regions to functional domains.

Comparative structural studies:

• Use homology modeling based on known crystal structures from related species.

• Identify species-specific structural elements that might confer unique functional properties.

• Map conservation onto 3D structures to identify functionally important residues.

Functional divergence assessment:

• Compare binding affinities to rRNA between different bacterial species.

• Assess interchangeability by complementation studies in different bacterial hosts.

• Evaluate differential responses to antibiotics targeting the ribosome.

This comparative approach provides insights into both fundamental ribosomal biology and species-specific features that might be exploited for targeted antibiotic development against Nocardia species while minimizing effects on beneficial bacteria.

What are the optimal conditions for expression and purification of recombinant N. farcinica rplC?

For researchers working with recombinant N. farcinica rplC, the following optimized protocol for expression and purification is recommended:

Expression optimization:

• Host selection: E. coli is the recommended expression host for rplC .

• Vector design: Incorporate a strong inducible promoter (T7 or tac) and appropriate fusion tags (His, GST, or MBP) to facilitate purification.

• Growth conditions:

  • Culture temperature: 25-30°C after induction (to reduce inclusion body formation)

  • Induction time: 4-6 hours with 0.5-1.0 mM IPTG

  • Media: Enriched media such as Terrific Broth supplemented with glucose

Purification strategy:

• Cell lysis: Sonication or high-pressure homogenization in a buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, and protease inhibitors.

• Initial capture: Affinity chromatography using the appropriate resin based on the fusion tag.

• Intermediate purification: Ion exchange chromatography to remove contaminants.

• Polishing: Size exclusion chromatography to obtain highly pure protein and remove aggregates.

• Quality control: SDS-PAGE analysis to confirm purity >85% .

Storage conditions:

• Short-term storage: 4°C for up to one week in an appropriate buffer.

• Long-term storage: Add glycerol to a final concentration of 5-50% and store at -20°C/-80°C.

• Lyophilization: For extended shelf life (up to 12 months) .

Researchers should avoid repeated freeze-thaw cycles as this can lead to protein degradation and loss of activity .

What analytical techniques are most effective for studying rplC interactions with rRNA and other ribosomal components?

Researchers investigating rplC interactions with rRNA and other ribosomal components should consider the following analytical approaches:

Biophysical interaction analysis:

• Surface Plasmon Resonance (SPR): Quantify binding kinetics between rplC and rRNA or other ribosomal proteins.

• Isothermal Titration Calorimetry (ITC): Determine thermodynamic parameters of binding.

• Microscale Thermophoresis (MST): Measure interactions in solution with minimal sample consumption.

• Bio-Layer Interferometry (BLI): Monitor real-time association and dissociation.

Structural analysis techniques:

• X-ray Crystallography: Determine high-resolution structures of rplC alone or in complex with binding partners.

• Cryo-Electron Microscopy: Visualize rplC within the context of the assembled ribosome.

• Nuclear Magnetic Resonance (NMR): Identify specific interaction surfaces for smaller domains.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Map protein-protein or protein-RNA interfaces.

Functional analysis methods:

• In vitro Translation Assays: Assess the impact of rplC mutations on protein synthesis.

• Ribosome Profiling: Analyze the effect of rplC variants on translation efficiency in vivo.

• Chemical Cross-linking coupled with Mass Spectrometry (XL-MS): Identify spatial relationships between rplC and other ribosomal components.

These complementary approaches provide a comprehensive understanding of how rplC contributes to ribosome assembly and function, potentially revealing novel targets for antibiotic development.

How can researchers differentiate between the roles of rplC and other ribosomal proteins in Nocardia farcinica antibiotic resistance?

To distinguish the specific contributions of rplC from other ribosomal proteins in N. farcinica antibiotic resistance, researchers should implement the following methodological approaches:

Genetic manipulation strategies:

• Gene knockout/knockdown: Create conditional mutants of rplC and other ribosomal protein genes.

• Complementation studies: Express wild-type or mutant rplC in knockout strains to assess functional rescue.

• Domain swapping: Create chimeric proteins between rplC and other ribosomal proteins to identify functional domains.

• Site-directed mutagenesis: Target specific residues predicted to be involved in antibiotic interactions.

Phenotypic analysis:

• Antimicrobial susceptibility testing: Compare minimum inhibitory concentrations (MICs) across mutant strains.

• Growth curve analysis: Assess fitness costs associated with mutations in different ribosomal proteins.

• Biofilm formation assays: Determine if alterations in ribosomal proteins affect biofilm development, which can contribute to antibiotic tolerance.

Molecular analysis:

• RNA-Seq: Profile transcriptional changes in response to antibiotic stress across different ribosomal protein mutants.

• Ribosome footprinting: Analyze changes in translation patterns and ribosome pausing.

• Proteomics: Identify changes in the protein composition of ribosomes in different mutant backgrounds.

Structural studies:

• Cryo-EM: Visualize structural changes in ribosomes due to mutations in different ribosomal proteins.

• In silico modeling: Predict and compare the effects of mutations on antibiotic binding sites.

By systematically applying these approaches, researchers can delineate the specific contributions of rplC to antibiotic resistance mechanisms in N. farcinica, potentially identifying novel targets for antimicrobial development.

What are common challenges in expressing soluble rplC protein and how can they be overcome?

Researchers frequently encounter challenges when expressing soluble recombinant N. farcinica rplC. Here are the common problems and solution strategies:

Challenge 1: Inclusion body formation
Solutions:

• Reduce expression temperature to 16-20°C after induction.

• Use lower inducer concentrations (0.1-0.2 mM IPTG).

• Express as fusion proteins with solubility-enhancing tags (MBP, SUMO, TrxA).

• Add osmolytes (sorbitol, glycine betaine) to the growth media.

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ).

Challenge 2: Proteolytic degradation
Solutions:

• Use protease-deficient E. coli strains (BL21, Rosetta).

• Include protease inhibitor cocktails during purification.

• Optimize buffer conditions (pH, salt concentration) to minimize proteolysis.

• Reduce purification time by streamlining the protocol.

• Maintain samples at 4°C throughout the purification process.

Challenge 3: Poor yield
Solutions:

• Optimize codon usage for E. coli expression.

• Use strong promoters and high-copy-number plasmids.

• Supplement growth media with rare amino acids or tRNAs.

• Screen multiple E. coli strains to identify optimal expression host.

• Consider auto-induction media for higher cell density and protein yield.

Challenge 4: Protein misfolding
Solutions:

• Include additives in purification buffers (glycerol, non-detergent sulfobetaines).

• Implement on-column refolding during affinity purification.

• Use mild detergents (0.1% Triton X-100) to stabilize hydrophobic regions.

• Add reducing agents (DTT, β-mercaptoethanol) to prevent disulfide scrambling.

• Perform buffer optimization screens to identify stabilizing conditions.

These strategies should be systematically tested to identify optimal conditions for producing high-quality soluble rplC protein for subsequent functional and structural studies.

How can researchers design experiments to investigate rplC's role in ribosome assembly and function?

To investigate rplC's role in ribosome assembly and function, researchers should design comprehensive experiments addressing multiple aspects of ribosomal biology:

In vitro ribosome assembly assays:

• Reconstitution experiments: Purify individual ribosomal components and assemble them in a step-wise manner.

  • Methodology: Mix purified rplC with 23S rRNA and monitor binding using filter binding assays or electrophoretic mobility shift assays.

  • Analysis: Determine binding constants and cooperative effects with other ribosomal proteins.

• Assembly kinetics: Monitor the time course of ribosome assembly with and without rplC.

  • Methodology: Use sucrose gradient centrifugation or light scattering to track assembly intermediates.

  • Analysis: Identify rate-limiting steps and assembly bottlenecks.

Structure-function studies:

• Mutagenesis approach: Create a library of rplC mutants targeting conserved residues.

  • Methodology: Express mutant proteins and assess their ability to incorporate into ribosomes.

  • Analysis: Map functional domains essential for assembly and activity.

• Domain deletion strategy: Remove specific structural elements from rplC.

  • Methodology: Express truncated versions and test their function in assembly assays.

  • Analysis: Identify minimal functional domains required for ribosome incorporation.

Functional assays:

• In vitro translation: Reconstitute ribosomes with wild-type or mutant rplC.

  • Methodology: Measure translation efficiency using reporter systems.

  • Analysis: Correlate structural features with translation activity.

• Antibiotic sensitivity: Test how rplC variants affect ribosome sensitivity to different antibiotics.

  • Methodology: Perform in vitro translation assays in the presence of various antibiotics.

  • Analysis: Identify rplC residues involved in antibiotic resistance or sensitivity.

In vivo approaches:

• Complementation studies: Express N. farcinica rplC in E. coli with conditional rplC mutations.

  • Methodology: Assess growth recovery under restrictive conditions.

  • Analysis: Determine functional conservation across species.

• Ribosome profiling: Analyze translation patterns with wild-type or mutant rplC.

  • Methodology: Sequence ribosome-protected mRNA fragments.

  • Analysis: Identify translation defects associated with rplC mutations.

These experimental designs provide a comprehensive framework for understanding rplC's fundamental role in ribosome biology and potential applications in antibiotic development.

What are the future research directions for studying N. farcinica rplC in the context of antibiotic resistance?

Future research on N. farcinica rplC should focus on several promising directions that could advance our understanding of ribosomal biology and antibiotic resistance mechanisms:

By pursuing these research directions, scientists can develop a more comprehensive understanding of how rplC contributes to N. farcinica biology and pathogenesis, potentially leading to new strategies for combating this clinically significant pathogen.

How does understanding rplC function contribute to broader knowledge of bacterial translation and antibiotic mechanisms?

The study of N. farcinica rplC contributes significantly to our broader understanding of bacterial translation and antibiotic mechanisms through several important perspectives:

• Evolutionary insights:

  • As a member of the universal ribosomal protein uL3 family , rplC represents a highly conserved component of the translation machinery.

  • Comparative studies between N. farcinica rplC and homologs from other bacteria reveal evolutionary constraints on ribosomal protein structure and function.

  • Species-specific variations highlight adaptations that may contribute to ecological niche specialization or pathogenesis.

• Structural biology advances:

  • rplC's role in nucleating 50S subunit assembly provides a model for understanding hierarchical assembly of large ribonucleoprotein complexes.

  • The protein's interactions with 23S rRNA reveal principles of RNA-protein recognition essential for ribosome function.

  • Structural studies of rplC contribute to our understanding of how the ribosome's catalytic center is organized and maintained.

• Antibiotic development implications:

  • Understanding rplC's structure and function can guide the development of new antibiotics that specifically target this essential protein.

  • Knowledge of how existing antibiotics interact with the ribosome can be leveraged to design derivatives with improved properties.

  • Insights from N. farcinica, which demonstrates resistance to several antibiotics , may reveal novel resistance mechanisms applicable to other pathogens.

• Fundamental translation mechanisms:

  • Research on rplC illuminates basic principles of translation fidelity, efficiency, and regulation.

  • Studies of how rplC mutations affect ribosome function provide insights into the molecular basis of protein synthesis.

  • The protein's conservation across species underscores its fundamental importance in the central dogma of molecular biology.

• Methodological advances:

  • Techniques developed for studying rplC can be applied to other ribosomal proteins and large macromolecular complexes.

  • Recombinant expression and purification protocols for rplC advance our ability to reconstitute ribosomes in vitro.

  • Structural and functional assays for rplC contribute to the toolkit available for studying translation across diverse bacterial species.