Recombinant Vibrio vulnificus 50S ribosomal protein L31 type B (rpmE2)

What is the structural composition of Vibrio vulnificus 50S ribosomal protein L31 type B (rpmE2)?

Vibrio vulnificus 50S ribosomal protein L31 type B (rpmE2) is a small ribosomal protein that forms part of the large 50S subunit in bacterial ribosomes. Based on homology modeling studies, the protein structure consists of 11 beta sheets and 2 alpha helices, creating a compact tertiary structure . This structural arrangement is consistent with its role in stabilizing the architecture of the large ribosomal subunit.

The protein shares approximately 33% sequence identity with its Escherichia coli homolog, which has been used as a template (PDB ID 2AW4) for structural modeling . The relatively modest sequence identity suggests structural conservation of key functional domains while allowing for species-specific adaptations.

How does rpmE2 differ from rpmE1 (L31 type A) in bacterial ribosomes?

The most significant distinction between L31 type B (rpmE2) and L31 type A (rpmE1) proteins lies in their zinc-binding capabilities:

Type B L31 proteins like rpmE2 are often expressed under zinc-limited conditions as a zinc-independent alternative to the zinc-requiring L31 type A proteins. This represents an adaptive mechanism allowing Vibrio vulnificus to maintain ribosomal function even during zinc starvation.

What is the genetic organization of the rpmE2 gene in the Vibrio vulnificus genome?

The rpmE2 gene in Vibrio vulnificus is located within a genomic context that reflects its functional role in zinc homeostasis. Unlike many housekeeping ribosomal protein genes that are organized in conserved operons, rpmE2 is often found in proximity to other zinc-regulated genes.

For effective characterization of the genomic context:

• Perform whole genome sequencing of your Vibrio vulnificus strain

• Use bioinformatic tools like BLAST and genome browsers to identify the exact location

• Analyze flanking sequences for regulatory elements, particularly Zur-binding motifs

• Compare syntenic regions across different Vibrio species to understand evolutionary conservation

What expression systems are most effective for recombinant production of Vibrio vulnificus rpmE2?

Based on research with similar ribosomal proteins, the following expression systems are recommended for recombinant rpmE2 production:

Methodology for optimal expression:

• Clone the rpmE2 gene into a vector with appropriate fusion tags (His6, MBP, or GST)

• Optimize expression conditions including temperature (16-37°C), inducer concentration, and duration

• Perform small-scale expression tests before scaling up

• Monitor protein expression using SDS-PAGE and Western blotting

• Evaluate protein solubility through fractionation experiments

Lowering the expression temperature to 16-18°C often improves solubility for ribosomal proteins that tend to aggregate at higher temperatures.

How can metal ion interactions with rpmE2 be experimentally characterized?

As a mercury-responsive protein , rpmE2's interactions with metal ions are critical to understanding its function. The following methodological approaches are recommended:

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding thermodynamics (Kd, ΔH, ΔS)

  • Requires 1-2 mg of purified protein

  • Protocol should include buffer matching and control experiments

• Differential Scanning Fluorimetry (DSF):

  • Measures thermal stability changes upon metal binding

  • Requires SYPRO Orange dye and real-time PCR equipment

  • Test multiple metal ions (Zn2+, Hg2+, Cd2+, Fe2+) at varying concentrations

• Circular Dichroism (CD) Spectroscopy:

  • Detects structural changes upon metal binding

  • Perform wavelength scans (190-260 nm) before and after metal addition

  • Monitor temperature-dependent unfolding to assess stability changes

• Inductively Coupled Plasma Mass Spectrometry (ICP-MS):

  • Quantifies metal content in purified protein samples

  • Requires acid digestion of protein samples

  • Essential for determining stoichiometry of binding

When studying mercury responsiveness specifically, safety protocols for handling mercury compounds must be strictly followed, including proper waste disposal procedures.

What are the optimal conditions for structural studies of rpmE2?

For high-resolution structural characterization of rpmE2:

X-ray Crystallography Approach:

• Purify protein to >95% homogeneity using multi-step chromatography

• Screen crystallization conditions using commercial kits at 4°C and 20°C

• Based on homology models showing 11 beta sheets and 2 alpha helices , focus on conditions successful for other beta-sheet-rich proteins

• Optimize promising conditions by varying precipitant concentration, pH, and protein concentration

• Consider addition of stabilizing additives like glycerol or specific metal ions

NMR Spectroscopy Approach:

• Express protein with 15N and 13C labeling in minimal media

• Purify to homogeneity and concentrate to 0.5-1.0 mM

• Optimize buffer conditions (typically 20 mM phosphate, pH 6.5-7.0, 50-150 mM NaCl)

• Perform HSQC experiments to assess sample quality before proceeding to 3D experiments

• For a protein with 11 beta sheets and 2 alpha helices , anticipate challenges with spectral crowding

Cryo-EM as an Alternative:
While traditionally challenging for small proteins, recent advances in Cryo-EM may allow visualization of rpmE2 in the context of the entire ribosome, providing insights into its structural position and interactions.

How does rpmE2 expression change during Vibrio vulnificus infection?

Understanding the expression patterns of rpmE2 during infection requires methodologies that can capture gene expression in complex host environments:

• RNA-Seq Analysis:

  • Extract RNA from Vibrio vulnificus grown in infection-mimicking conditions (low iron, temperature shifts, host cell contact)

  • Compare with expression in standard laboratory conditions

  • Use DESeq2 or similar statistical tools to identify differential expression

  • Validate findings with RT-qPCR

• In vivo Expression Technology (IVET):

  • Create promoter-reporter constructs for rpmE2

  • Infect appropriate animal models

  • Recover bacteria from different infection sites

  • Measure reporter activity to determine spatial and temporal expression patterns

• Dual RNA-Seq:

  • Simultaneously profile bacterial and host transcriptomes during infection

  • Identify correlation between rpmE2 expression and host response genes

  • Map expression changes to specific infection stages

Vibrio vulnificus is highly lethal with mortality rates exceeding 50% in systemic infections . Understanding how ribosomal proteins like rpmE2 contribute to survival in the host environment could reveal new insights into virulence mechanisms.

Can rpmE2 serve as a target for novel antimicrobial agents against Vibrio vulnificus?

To evaluate rpmE2 as a potential antimicrobial target:

• Target Validation:

  • Generate conditional knockdown strains using antisense RNA or CRISPR interference

  • Assess growth phenotypes in standard and infection-mimicking conditions

  • Evaluate impact on virulence in cell culture and animal models

  • Determine essentiality under different environmental conditions

• Structural Analysis for Drug Design:

  • Use the homology model showing 11 beta sheets and 2 alpha helices to identify potential binding pockets

  • Perform molecular dynamics simulations to identify flexible regions

  • Apply fragment-based screening approaches

  • Design peptidomimetics that can disrupt ribosome assembly

• High-Throughput Screening:

  • Develop assays measuring ribosome assembly or function

  • Screen compound libraries for inhibitors

  • Validate hits with secondary assays (binding, antimicrobial activity)

  • Assess specificity by testing against human ribosomes

Given Vibrio vulnificus's high lethality , novel antimicrobial approaches targeting ribosomal proteins could provide valuable therapeutic options, particularly for antibiotic-resistant strains.

What is the role of rpmE2 in zinc homeostasis in Vibrio vulnificus?

The role of rpmE2 in zinc homeostasis involves complex regulatory mechanisms:

• Zinc-Responsive Expression:

  • Typically, L31 type B proteins are expressed during zinc limitation

  • Measure rpmE2 expression using RT-qPCR under varying zinc concentrations

  • Determine if expression is regulated by the zinc uptake regulator (Zur)

  • Identify Zur binding sites in the rpmE2 promoter region

• Functional Replacement:

  • Determine if rpmE2 can functionally replace rpmE1 in zinc-limited conditions

  • Generate single and double knockout strains

  • Assess growth phenotypes and ribosome profiles

  • Measure translation efficiency using reporter systems

• Zinc Mobilization:

  • Investigate whether rpmE2 expression coincides with release of zinc from other zinc-binding proteins

  • Measure intracellular zinc levels using zinc-specific fluorescent probes

  • Monitor zinc redistribution during stress responses

The zinc-responsive expression of L31 type B proteins represents an elegant adaptive mechanism that allows bacteria to maintain ribosomal function while redirecting zinc to essential processes during limitation.

How does mercury stress influence rpmE2 expression and function?

Mercury stress significantly impacts rpmE2, as it has been identified as a mercury-responsive protein :

• Expression Analysis:

  • Expose Vibrio vulnificus cultures to sub-lethal mercury concentrations

  • Measure rpmE2 transcript levels by RT-qPCR at different time points

  • Perform Western blot analysis to confirm protein-level changes

  • Compare with other mercury-responsive genes to identify co-regulation patterns

• Functional Impact:

  • Assess ribosome integrity and composition after mercury exposure

  • Measure translation efficiency using reporter systems

  • Determine if mercury directly binds to rpmE2 using ICP-MS

  • Investigate structural changes using CD spectroscopy

• Protective Mechanisms:

  • Generate rpmE2 knockout strains and assess mercury sensitivity

  • Complementation studies with wild-type and mutant rpmE2

  • Investigate interactions with other mercury stress response pathways

Understanding mercury responsiveness of rpmE2 may reveal novel mechanisms for metal detoxification and adaptation to environmental stressors.

What are the best computational approaches for homology modeling of rpmE2?

For accurate homology modeling of Vibrio vulnificus rpmE2:

• Template Selection:

  • Based on existing research, the E. coli ribosome structure (PDB ID 2AW4) provides a suitable template with 33% sequence identity

  • Search for additional templates using HHpred or SWISS-MODEL

  • Consider multiple templates if available to improve model accuracy

• Alignment Optimization:

  • Generate multiple sequence alignments of L31 proteins across bacterial species

  • Manually refine alignments focusing on conserved secondary structure elements

  • Pay special attention to the positioning of the 11 beta sheets and 2 alpha helices identified in previous models

• Model Building and Refinement:

  • Use software such as MODELLER, SWISS-MODEL, or Rosetta for initial model building

  • Refine models using molecular dynamics simulations

  • Validate models using PROCHECK, VERIFY3D, and ERRAT

  • Generate multiple models and select the best based on quality metrics

• Functional Site Prediction:

  • Identify potential metal binding sites

  • Map conservation onto the structural model

  • Predict protein-protein interaction interfaces

  • Identify conformationally flexible regions

The resulting model should accurately represent the arrangement of the 11 beta sheets and 2 alpha helices characteristic of this protein .

How can molecular dynamics simulations enhance our understanding of rpmE2 function?

Molecular dynamics (MD) simulations offer powerful insights into rpmE2 behavior:

• Simulation Setup:

  • Use the homology model based on the E. coli ribosome template (PDB ID 2AW4)

  • Solvate in explicit water with physiological ion concentrations

  • Apply CHARMM36 or AMBER ff19SB force fields

  • Include parameters for metal ions if studying metal binding

• Analysis Approaches:

  • Calculate root-mean-square deviation (RMSD) and fluctuation (RMSF)

  • Analyze secondary structure stability, focusing on the 11 beta sheets and 2 alpha helices

  • Identify allosteric communication pathways

  • Perform principal component analysis to identify dominant motions

• Specific Simulations:

  • Compare dynamics in presence/absence of zinc or mercury

  • Simulate interaction with rRNA and neighboring proteins

  • Perform free energy calculations for metal binding

  • Study conformational changes during ribosome assembly

• Integration with Experimental Data:

  • Validate simulation findings against experimental measurements

  • Use simulation to interpret ambiguous experimental results

  • Design targeted mutations based on simulation predictions

MD simulations complement experimental approaches by providing atomic-level details of dynamic processes that are challenging to capture experimentally.

How can contradictory findings about rpmE2 function be reconciled?

Resolving contradictory findings requires systematic methodological approaches:

• Contextual Analysis:

  • Compare experimental conditions (growth media, temperature, stress conditions)

  • Assess strain differences (clinical vs. environmental isolates)

  • Evaluate methodological differences (in vitro vs. in vivo approaches)

  • Consider temporal factors (growth phase, duration of stress exposure)

• Integrative Approaches:

  • Combine multiple experimental techniques to address the same question

  • Perform meta-analysis of available data

  • Use computational models to simulate different experimental conditions

  • Design experiments that can directly test competing hypotheses

• Systematic Validation:

  • Replicate key experiments using standardized protocols

  • Perform dose-response or time-course studies to capture dynamics

  • Use genetic approaches (knockouts, complementation, point mutations)

  • Employ biochemical methods to isolate specific interactions

For rpmE2 specifically, contradictions might arise from differences in metal availability or ribosome assembly conditions that affect protein behavior.

What quality control measures should be implemented when studying recombinant rpmE2?

Rigorous quality control ensures reliable research outcomes:

• Protein Quality Assessment:

  • Verify protein identity by mass spectrometry

  • Assess purity using SDS-PAGE and size exclusion chromatography

  • Confirm proper folding using circular dichroism

  • Measure activity through functional assays

• Experimental Controls:

  • Include wild-type controls in all experiments

  • Use inactive mutants as negative controls

  • Perform parallel experiments with related proteins (e.g., rpmE1)

  • Include technical and biological replicates

• Data Validation:

  • Apply appropriate statistical tests

  • Use multiple independent methods to verify key findings

  • Implement blinding procedures where applicable

  • Share raw data and detailed protocols

• Reproducibility Measures:

  • Document all experimental conditions in detail

  • Maintain consistent sources of reagents and materials

  • Standardize protocols across different researchers

  • Implement laboratory information management systems

Special considerations for rpmE2 include monitoring metal content throughout purification and ensuring ribosomal context is appropriately maintained or reconstituted for functional studies.