Recombinant Erwinia carotovora subsp. atroseptica UPF0283 membrane protein ECA1987 (ECA1987)

What is ECA1987 and what organism does it originate from?

ECA1987 is a UPF0283 family membrane protein consisting of 347 amino acids. It originates from Erwinia carotovora subsp. atroseptica (currently reclassified as Pectobacterium atrosepticum), a gram-negative plant pathogenic bacterium that causes blackleg and soft rot in potatoes and other crops . The protein has the UniProt ID Q6D5Q4 and contains multiple transmembrane domains that anchor it within bacterial cell membranes .

How is recombinant ECA1987 typically produced for research?

Recombinant ECA1987 is typically produced in E. coli expression systems with an N-terminal His-tag fusion to facilitate purification. The protein is expressed in E. coli under controlled conditions and then isolated through affinity chromatography leveraging the His-tag . After purification, the protein is often prepared as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 to maintain stability during storage . This production approach allows researchers to obtain sufficient quantities of pure protein for structural and functional studies.

What are the proper storage conditions for recombinant ECA1987?

Recombinant ECA1987 should be stored at -20°C to -80°C upon receipt. For long-term storage, it is recommended to add glycerol (final concentration 30-50%) and create aliquots to avoid repeated freeze-thaw cycles, which can compromise protein integrity . When working with the protein, temporary storage of working aliquots at 4°C for up to one week is acceptable . Prior to opening, vials should be briefly centrifuged to bring contents to the bottom.

What quality control mechanisms affect membrane protein folding of proteins like ECA1987?

Membrane proteins such as ECA1987 are subject to rigorous quality control mechanisms during synthesis and folding. Research indicates that eukaryotic cells employ a pre-emptive quality control pathway for membrane proteins that fail to properly assemble in the endoplasmic reticulum (ER) . The ER membrane complex (EMC) plays a crucial role in this process, and its absence can lead to reduced synthesis of misfolded membrane proteins .

For bacterial membrane proteins like ECA1987, similar quality control mechanisms exist, though with variations appropriate to prokaryotic cell architecture. When membrane domain insertion or folding fails, ribosome-associated quality control pathways are triggered by ribosome collisions . This mechanism serves to prevent the accumulation of potentially toxic aggregation-prone proteins within the membrane environment.

How do ribosomal dynamics influence the expression and folding of ECA1987?

Research on membrane proteins has revealed that ribosomal dynamics significantly influence both expression and proper folding. For complex membrane proteins like ECA1987, evidence suggests that translational tuning occurs to balance protein synthesis and folding rates . Studies across the yeast membrane proteome have demonstrated that polytopic membrane proteins typically exhibit relatively low ribosome abundance, which supports the hypothesis that slower translation rates may facilitate proper membrane insertion and folding .

For ECA1987 specifically, optimizing translation initiation and elongation rates during recombinant expression can be critical for obtaining properly folded protein. Ribosomal mutations or manipulations that reduce ribosome abundance on the message have been shown to rescue synthesis defects in some membrane proteins . This suggests that controlling translation kinetics could be a valuable approach for improving the expression of functional ECA1987.

What structural motifs in ECA1987 are most critical for its membrane integration?

Based on the amino acid sequence analysis of ECA1987, several hydrophobic regions likely form transmembrane domains that are essential for membrane integration. The sequence "RWRRMVMAGVALFGISALAQGVQS" and similar hydrophobic stretches are predicted to be critical for proper membrane insertion and topology .

Computational analysis suggests ECA1987 contains multiple transmembrane helices that anchor it in the bacterial membrane. Mutations in these regions would likely disrupt membrane integration and consequently protein function. Advanced structural studies utilizing circular dichroism, NMR, or X-ray crystallography would be necessary to fully elucidate which specific structural motifs are most critical for membrane integration and function.

What expression system optimizations are recommended for high-yield production of functional ECA1987?

When designing expression systems for ECA1987, careful consideration must be given to the translational tuning required for proper membrane protein folding. Evidence suggests that slowing translation can improve membrane protein folding outcomes . Therefore, using lower temperatures (16-18°C) and reduced inducer concentrations can facilitate proper insertion into membranes. Additionally, specialized E. coli strains like C41(DE3), which are adapted for toxic membrane protein expression, might yield better results than standard strains.

How can researchers assess proper folding and membrane integration of recombinant ECA1987?

Assessing proper folding and membrane integration of ECA1987 requires a multi-method approach:

• Membrane Fractionation Analysis: Separation of properly integrated protein in membrane fractions versus misfolded protein in inclusion bodies through differential centrifugation and Western blotting.

• Protease Protection Assays: Limited proteolysis of membrane vesicles containing ECA1987 to determine which domains are protected (integrated into the membrane) versus exposed (accessible to proteases).

• Fluorescence-Based Folding Assays: Introduction of site-specific fluorescent labels to monitor conformational states and folding kinetics.

• Circular Dichroism Spectroscopy: Assessment of secondary structure content to confirm proper folding compared to predicted structural elements.

These complementary approaches provide different perspectives on protein folding and membrane integration, allowing researchers to comprehensively evaluate ECA1987's structural integrity.

What are the recommended reconstitution protocols for lyophilized ECA1987 protein?

For optimal reconstitution of lyophilized ECA1987, the following step-by-step protocol is recommended:

• Centrifuge the vial briefly (30 seconds at 10,000 g) to collect the lyophilized powder at the bottom.

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL, mixing gently until completely dissolved .

• For long-term storage, add glycerol to a final concentration of 30-50% (optimal concentration is 50%) .

• Create small working aliquots to minimize freeze-thaw cycles.

• For functional studies, consider buffer exchange into a more physiologically relevant buffer using dialysis or size exclusion chromatography.

• Verify protein integrity post-reconstitution via SDS-PAGE analysis.

This protocol maintains protein stability while minimizing aggregation, which is particularly important for membrane proteins that tend to aggregate in aqueous solutions.

How should researchers address potential data inconsistencies when studying ECA1987 function?

When encountering data inconsistencies in ECA1987 functional studies, researchers should systematically evaluate several factors:

• Protein Quality Assessment: Verify protein purity (>90% by SDS-PAGE) and integrity after purification and storage . Aggregated or degraded protein can produce inconsistent results.

• Membrane Environment Variations: Document and standardize lipid composition in reconstitution experiments, as membrane proteins are highly sensitive to their lipid environment.

• Post-translational Modifications: Evaluate potential differences in post-translational modifications between native and recombinant proteins, which may affect function.

• Experimental Conditions Matrix: Create a comprehensive matrix of experimental conditions (pH, temperature, ionic strength) to identify variables that contribute to inconsistencies.

• Technical Replicates vs. Biological Replicates: Distinguish between technical variability (same protein preparation, multiple measurements) and biological variability (different protein preparations).

By systematically addressing these factors, researchers can identify sources of variability and establish more consistent experimental conditions for studying ECA1987.

What comparative approaches are most informative when studying UPF0283 family proteins across bacterial species?

When comparing UPF0283 family proteins like ECA1987 across bacterial species, researchers should employ multiple complementary approaches. Sequence alignment reveals conserved motifs that may be critical for function, while structural modeling can predict three-dimensional similarities despite sequence divergence. Expression pattern analysis across different bacterial species and environmental conditions can reveal functional conservation or specialization.

The most informative approach combines computational phylogenetic analysis with experimental cross-species complementation studies, where the ability of one species' protein to functionally replace another is directly tested.

How can researchers effectively study membrane protein interactions involving ECA1987?

To effectively study potential protein-protein interactions involving ECA1987, researchers should consider these specialized techniques for membrane proteins:

• Membrane Yeast Two-Hybrid (MYTH): A modified yeast two-hybrid system specifically designed for membrane proteins that allows detection of interactions in a membrane environment.

• Bimolecular Fluorescence Complementation (BiFC): Fusion of potential interaction partners with complementary fragments of a fluorescent protein to visualize interactions in living cells.

• Crosslinking Mass Spectrometry: Chemical crosslinking followed by mass spectrometry analysis to identify interaction interfaces in native membrane environments.

• Co-immunoprecipitation with Membrane-Compatible Detergents: Using mild detergents that maintain membrane protein structure while solubilizing complexes.

• Surface Plasmon Resonance (SPR): For in vitro quantification of binding kinetics between purified ECA1987 and potential interaction partners in a lipid bilayer environment.

These techniques address the unique challenges of studying membrane protein interactions, which are difficult to capture with conventional protein interaction methods due to the hydrophobic nature of membrane proteins.

How might ECA1987 function relate to Erwinia carotovora pathogenicity in plant hosts?

Understanding the potential role of ECA1987 in pathogenicity requires considering the ecological context of Erwinia carotovora (Pectobacterium atrosepticum). This pathogen is known to cause blackleg and aerial stem soft rot in potatoes and can be transmitted through irrigation water . While there isn't direct evidence linking ECA1987 specifically to pathogenicity in the provided search results, membrane proteins often play crucial roles in bacteria-host interactions.

Research approaches to investigate this connection should include:

• Gene knockout or knockdown studies to observe effects on virulence

• Expression analysis during different stages of plant infection

• Localization studies during host interaction

• Comparative analysis of ECA1987 homologs across pathogenic and non-pathogenic bacterial strains

The presence of Erwinia carotovora in surface and well water sources in agricultural regions suggests potential environmental reservoirs for this pathogen , making the study of its membrane proteins relevant to understanding transmission dynamics and developing control strategies.

What structural biology techniques are most promising for determining the 3D structure of ECA1987?

For ECA1987, a combined approach may be most effective: using computational prediction with AlphaFold2 as a starting point, followed by experimental validation using either cryo-EM or X-ray crystallography depending on the protein's stability in detergent solutions versus lipidic cubic phase environments. This multi-technique strategy addresses the inherent challenges of membrane protein structural biology.