Recombinant Rhodopirellula baltica Elongation factor 4 1 (lepA1), partial

What is Elongation factor 4 1 (lepA1) and what is its role in Rhodopirellula baltica?

Elongation factor 4 1 (lepA1), also known as EF-4 1 or Ribosomal back-translocase LepA 1 (EC 3.6.5.n1), is a GTPase involved in protein synthesis in Rhodopirellula baltica . As a member of the translational GTPase family, lepA1 likely contributes to translation fidelity by catalyzing back-translocation of mispositioned tRNAs during protein synthesis. Notably, R. baltica contains this specialized elongation factor that may be adapted to its unique ecological niche as an attached-living marine bacterium found in brackish waters .

Unlike the more extensively studied EF-Tu and EF-G, lepA1 appears to function primarily under stress conditions, helping the organism maintain translation accuracy when exposed to environmental challenges typical in marine environments.

How does lepA1 from R. baltica compare to homologous proteins in other bacteria?

While the specific sequence homology data for R. baltica lepA1 is limited in the search results, research indicates that this marine bacterium possesses unique adaptations in its translational machinery. Similar to its membrane insertases with extended C-terminal regions , lepA1 likely contains structural features adapted to the marine environment.

The unique adaptations observed in R. baltica proteins suggest that lepA1 likely contains molecular features tailored to the organism's ecology in the Baltic Sea and other European marine environments .

What expression systems are optimal for producing recombinant R. baltica lepA1?

Multiple expression systems have been employed for recombinant production of R. baltica lepA1, each with specific advantages :

For functional studies requiring properly folded protein with minimal modifications, the E. coli system typically provides sufficient quality with optimal yield. For structural studies or when authentic post-translational modifications are crucial, insect or mammalian expression systems may be preferable despite lower yields.

What purification strategies yield the highest purity and activity of recombinant lepA1?

Based on standard protocols for GTPases and the information available for recombinant R. baltica proteins , the following purification workflow is recommended:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using the His-tag (or appropriate fusion tag)

• Intermediate purification: Ion exchange chromatography to separate charge variants

• Polishing step: Size exclusion chromatography to remove aggregates and ensure monodispersity

Critical considerations for maintaining activity:

• Include GTP or GDP (1-5 μM) in all buffers to stabilize the protein

• Maintain 5-10 mM MgCl₂ throughout purification

• Consider adding glycerol (10-20%) to prevent aggregation

• Perform quality control using SDS-PAGE (target purity >85%)

• Verify activity using GTPase assays before and after each purification step

The final product should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with optional addition of 5-50% glycerol as a cryoprotectant for long-term storage .

How can researchers design robust assays to measure lepA1 GTPase activity?

The GTPase activity of lepA1 can be assessed through multiple complementary approaches:

Colorimetric phosphate detection assay:

• Incubate purified lepA1 (0.1-1 μM) with GTP (50-200 μM) in buffer containing 50 mM Tris-HCl pH 7.5, 70 mM NH₄Cl, 30 mM KCl, and 7 mM MgCl₂ at 30°C

• At timed intervals (0-60 min), remove aliquots and measure released phosphate using malachite green reagent

• Calculate initial rates to determine specific activity (nmol Pi/min/mg protein)

Coupled-enzyme assay for continuous monitoring:

• Link GTP hydrolysis to NADH oxidation via pyruvate kinase and lactate dehydrogenase

• Monitor decrease in A₃₄₀ as NADH is consumed

• Calculate rates based on the extinction coefficient of NADH

Controls and variations:

• Include ribosome-stimulated activity measurements (using purified ribosomes at 0.1-0.5 μM)

• Test activity across temperature range (4-37°C) and pH range (6.0-8.5)

• Assess the effect of salt concentration to model brackish water conditions

• Compare activity with and without potential regulatory factors

What approaches can be used to study the interaction between lepA1 and ribosomes?

The lepA1-ribosome interaction can be characterized using several biophysical and biochemical methods:

Co-sedimentation assays:

• Incubate purified lepA1 with ribosomes from R. baltica or heterologous sources

• Subject the mixture to ultracentrifugation through sucrose cushion

• Analyze pellet and supernatant fractions by SDS-PAGE to determine binding

Surface Plasmon Resonance (SPR):

• Immobilize ribosomes on sensor chip

• Flow lepA1 at varying concentrations

• Determine association and dissociation rate constants

• Calculate binding affinity (KD)

Cryo-electron microscopy:
Based on the approach used for visualizing YidC-ribosome complexes from R. baltica , researchers can:

• Form stable complexes of lepA1 with ribosomes using non-hydrolyzable GTP analogs

• Vitrify samples and collect high-resolution images

• Perform 3D reconstruction to visualize the binding interface

• Target resolution of ~8-9 Å or better to identify key interaction points

The enhanced affinity observed with C-terminally extended YidC chimeras suggests that lepA1 may also have specialized regions that facilitate ribosome interaction. Characterizing these interactions could reveal adaptations specific to R. baltica's marine environment.

What structural features distinguish lepA1 from other translational GTPases?

While specific structural data for R. baltica lepA1 is limited in the search results, translational GTPases typically share core domains while having distinct features that confer specialized functions:

Researchers should consider:

• Performing comparative sequence analysis with LepA/EF4 from diverse bacteria

• Generating homology models based on solved structures of related proteins

• Conducting molecular dynamics simulations to predict behavior under various salt concentrations

• Pursuing X-ray crystallography or cryo-EM studies for definitive structural characterization

How might site-directed mutagenesis help elucidate structure-function relationships in lepA1?

Site-directed mutagenesis offers a powerful approach to probe specific aspects of lepA1 function:

Key residues to target:

• GTP-binding motifs (G1-G5) to disrupt nucleotide binding/hydrolysis

• Putative ribosome-binding interfaces based on homology modeling

• Conserved residues in the potential back-translocation domain

• Marine-specific residues identified through comparative sequence analysis

Experimental design:

• Generate a panel of point mutations using PCR-based mutagenesis

• Express and purify mutant proteins using the established workflow

• Conduct parallel assays comparing wild-type and mutant proteins:

  • Intrinsic and ribosome-stimulated GTPase activity

  • Ribosome binding affinity

  • Back-translocation activity

  • Stability under varying salt and temperature conditions

Expected outcomes:

• Identification of residues essential for catalysis versus ribosome binding

• Correlation between specific domains and functional activities

• Discovery of adaptations unique to marine planctomycetes

• Insight into evolutionary adaptations in the translational machinery

How might lepA1 contribute to R. baltica's adaptation to marine environments?

R. baltica is an attached-living marine bacterium found in brackish waters including the Baltic Sea . The lepA1 protein likely contributes to environmental adaptation through several mechanisms:

• Stress response: As a back-translocase, lepA1 may help maintain translation fidelity under osmotic stress conditions typical in brackish waters where salinity fluctuates

• Temperature adaptation: Given the broad distribution of Rhodopirellula species across European seas , lepA1 could facilitate protein synthesis across varying temperature ranges

• Biofilm formation: As an attached-living bacterium , R. baltica's translational machinery may be specialized for the physiological state of surface-attached growth, with lepA1 potentially playing a role in regulating translation during attachment

• Response to pollutants: R. baltica strains have been isolated from contaminated environments like Brazilian mangroves , suggesting potential lepA1 involvement in maintaining protein synthesis under xenobiotic stress

Research approaches to investigate these adaptations include:

• Comparative activity assays under varying salt concentrations mimicking different marine environments

• Temperature-dependent activity profiling

• Transcriptional analysis of lepA1 expression under various stress conditions

• Heterologous expression studies comparing R. baltica lepA1 with homologs from non-marine bacteria

What is the significance of having multiple expression systems available for lepA1 research?

The availability of lepA1 expressed in different systems (E. coli, yeast, baculovirus, mammalian) offers significant advantages for comprehensive characterization:

Complementary approaches for structural studies:

• E. coli-expressed protein for initial crystallization trials and NMR studies

• Insect cell-expressed protein for cryo-EM and more challenging crystallization

• Mammalian-expressed protein when authentic post-translational modifications are critical

Functional comparative analysis:

• Evaluate whether expression system affects activity or binding properties

• Identify potential post-translational modifications unique to eukaryotic expression

• Determine if folding environment influences structural stability

Specialized applications:

• Biotinylated protein (CSB-EP763336RDR-B) for surface immobilization in SPR or pull-down assays

• Yeast-expressed protein for eukaryotic interaction studies

• Higher purity preparations from mammalian cells for sensitive biophysical characterization

This range of options allows researchers to select the most appropriate preparation based on their specific experimental requirements and research questions.

How can comparative genomics inform our understanding of lepA1 evolution in Planctomycetes?

The Planctomycetes phylum, to which R. baltica belongs, represents one of the least explored bacterial groups despite their ability to survive in diverse environments . Comparative genomic approaches can reveal evolutionary insights about lepA1:

• Phylogenetic analysis: Construct phylogenetic trees based on lepA1 sequences from various Planctomycetes and other bacterial phyla to trace evolutionary history

• Gene neighborhood analysis: Examine genomic context of lepA1 across species to identify potential functional partners and operonic structures

• Selective pressure analysis: Calculate dN/dS ratios to identify regions under positive or purifying selection

• Horizontal gene transfer assessment: Evaluate whether lepA1 shows evidence of horizontal acquisition or vertical inheritance within Planctomycetes

The genetic diversity observed among Rhodopirellula isolates from European seas suggests that lepA1 may show regional variations that could be correlated with specific environmental conditions, providing insight into adaptation mechanisms.

What role might recombinant lepA1 play in studying R. baltica's unique cellular compartmentalization?

Planctomycetes like R. baltica exhibit unusual cellular organization with membrane-bound compartments that challenge traditional views of bacterial cell structure. Recombinant lepA1 could serve as a tool to investigate these unique features:

• Localization studies: Using fluorescently-labeled recombinant lepA1 to determine its subcellular distribution in R. baltica

• Membrane association: Investigating whether lepA1 interacts with specific membrane structures unique to Planctomycetes

• Compartment-specific translation: Exploring the possibility that protein synthesis machinery, including lepA1, may be differentially distributed across cellular compartments

• Evolutionary implications: Comparing lepA1 structure and function with homologs from bacteria lacking complex cellular compartmentalization

Such studies could provide fundamental insights into how essential cellular processes like translation have adapted to the unique cellular architecture of Planctomycetes.

What are the optimal storage conditions for maintaining lepA1 stability and activity?

Based on product information and standard practices for recombinant proteins:

For lyophilized protein:

• Store at -20°C to -80°C in sealed containers with desiccant

• Protect from moisture and repeated temperature fluctuations

• Typical shelf-life under these conditions: 12 months

For reconstituted protein:

• Short-term (2-7 days): 4-8°C

• Medium-term (up to 3 months): -20°C with 5-50% glycerol added as cryoprotectant

• Aliquot to avoid repeated freeze-thaw cycles

• Consider adding reducing agents (1-5 mM DTT) if the protein contains cysteines

Activity monitoring:

• Perform periodic GTPase activity assays to verify functional integrity

• Monitor for precipitation or turbidity prior to use

• Consider including stabilizing agents (GTP/GDP at 1-5 μM) for extended storage

What quality control measures should be implemented for recombinant lepA1 preparations?

A comprehensive quality control protocol for recombinant lepA1 should include:

Physical characterization:

• SDS-PAGE analysis (target: >85% purity)

• Western blot with anti-tag or anti-lepA1 antibodies

• Mass spectrometry to confirm intact mass and detect modifications

• Dynamic light scattering to assess monodispersity and aggregation status

Functional verification:

• Intrinsic GTPase activity (baseline level)

• Ribosome-stimulated GTPase activity (expected enhancement)

• Ribosome binding assay (affinity measurement)

• Thermal stability analysis (melting temperature determination)

Biological activity:

• In vitro translation assay to confirm functional impact on protein synthesis

• Back-translocation activity assessment if appropriate systems are available

• Comparison with positive control (e.g., E. coli LepA) when possible

Implementing these quality control measures ensures experimental reproducibility and allows meaningful comparison of results across different studies.