Recombinant Photobacterium profundum SsrA-binding protein (smpB)

What is the molecular function of SmpB in bacterial systems?

SmpB functions as an essential component of the bacterial SsrA quality-control system, which recognizes ribosomes stalled on defective mRNAs. The SmpB protein binds specifically and with high affinity to SsrA RNA (with an affinity of approximately 20 nM), enabling the SsrA RNA to act as both tRNA and mRNA to mediate the addition of a short peptide tag to the C-terminus of partially synthesized polypeptide chains . This SmpB-SsrA complex formation is critical for mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain . Importantly, SmpB is required for stable association of SsrA with ribosomes in vivo, as demonstrated by fractionation experiments showing that in SmpB-deficient cells, SsrA fails to co-sediment with 70S ribosomes .

What is the structural composition of Photobacterium profundum SmpB?

Photobacterium profundum SmpB (Uniprot No. Q6LUB4) is a full-length protein consisting of 158 amino acids with the sequence: MAKKPNKSDNTIAKNRTARHEFAIQDDYEAGLQLQGWEVKAIRNGKVNIAESYVFLRDGEAFISGVTITPLNAASTHVVADPTRTRKLLLNRKEIDKLLGAVNREGQTIVALSMYWKASWVKLKIGTARGKKLHDKRADSKSRDWARDKQRIMKHSTR . Circular dichroism studies of SmpB proteins indicate they are predominantly β-sheet proteins, although specific structural data for the P. profundum variant would require detailed crystallographic or NMR studies .

How should researchers store and reconstitute recombinant P. profundum SmpB?

For optimal stability, recombinant P. profundum SmpB should be briefly centrifuged prior to opening to bring contents to the bottom of the vial. Reconstitution should be performed in deionized sterile water to a concentration of 0.1-1.0 mg/mL, followed by the addition of glycerol to a final concentration of 5-50% (with 50% being the standard recommendation) . The reconstituted protein should be aliquoted to avoid repeated freeze-thaw cycles and stored at -20°C/-80°C . Liquid formulations typically have a shelf life of approximately 6 months at -20°C/-80°C, while lyophilized forms can remain stable for up to 12 months under the same storage conditions .

How can researchers assess the RNA-binding specificity of P. profundum SmpB?

To evaluate the RNA-binding specificity of P. profundum SmpB, researchers should consider employing gel-mobility shift assays using purified recombinant protein and labeled RNA. Based on established protocols with other SmpB proteins, binding assays should be conducted in buffer containing physiologically relevant salt concentrations (approximately 200 mM KCl) . For competition experiments to assess specificity, unlabeled SsrA RNA and total yeast tRNA can be used as competitors against labeled SsrA binding. A genuine SmpB-SsrA interaction should demonstrate significantly higher affinity for SsrA RNA compared to non-specific RNAs (approximately 400-fold higher affinity has been observed with E. coli SmpB) . Researchers should also consider using techniques such as isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) for more quantitative binding kinetics and thermodynamics measurements.

How does pressure affect SmpB function in Photobacterium profundum, and how can this be experimentally measured?

Given that Photobacterium profundum is a piezophilic (pressure-loving) bacterium isolated from the deep sea, the functionality of its SmpB protein may be adapted to high-pressure environments . To investigate pressure effects, researchers should consider:

• Comparative binding assays performed under different pressure conditions using high-pressure biophysical techniques

• MS-based label-free quantitative proteomics to assess changes in SmpB expression levels under different pressure conditions

• Ribosome association experiments conducted in pressure chambers to determine if SmpB-SsrA-ribosome interactions are pressure-dependent

Such analyses would require specialized equipment capable of maintaining samples under high-pressure conditions while performing biochemical assays. Results should be compared with SmpB proteins from non-piezophilic organisms to identify potential adaptations specific to deep-sea bacteria.

What experimental approaches can be used to investigate the role of SmpB in stress responses in P. profundum?

To investigate SmpB's role in stress responses, researchers should consider these methodological approaches:

• Create a clean smpB deletion mutant in P. profundum using allelic exchange techniques

• Compare phenotypes of wild-type and ΔsmpB strains under various stress conditions (temperature, pressure, nutrient limitation, antibiotics)

• Conduct RNA-seq analysis to identify transcriptome-wide changes resulting from smpB deletion

• Perform ribosome profiling to assess translation dynamics in the absence of SmpB

• Use quantitative proteomics to identify proteins whose levels are affected by smpB deletion

Based on studies in other bacteria, researchers should pay particular attention to phage development phenotypes and protein tagging defects, as these have been documented as characteristic outcomes of SmpB deficiency .

How can researchers validate the functionality of recombinant P. profundum SmpB?

Functional validation of recombinant P. profundum SmpB should include:

• RNA-binding assays: Gel-mobility shift assays using both homologous P. profundum SsrA RNA and heterologous SsrA RNAs to assess binding specificity and affinity

• Complementation assays: Introduction of the P. profundum smpB gene into an E. coli ΔsmpB strain to determine if it can restore:

  • SsrA-mediated tagging activity

  • Phage development phenotypes

  • Ribosome association of SsrA RNA

• Ribosome binding assays: Sucrose gradient fractionation experiments to assess whether the recombinant SmpB can facilitate SsrA RNA association with ribosomes in vitro or in vivo

• Circular dichroism spectroscopy: To confirm proper protein folding by comparing spectral characteristics with those of other characterized SmpB proteins

These assays collectively provide a robust assessment of whether the recombinant protein possesses the biochemical and biological activities expected of functional SmpB.

What are the critical controls needed when studying SmpB-SsrA interactions in P. profundum?

When designing experiments to study SmpB-SsrA interactions in P. profundum, researchers should include these essential controls:

• Non-specific RNA binding control: Include structured RNAs other than SsrA (such as tRNAs) to demonstrate binding specificity

• Salt concentration controls: Perform binding assays at multiple salt concentrations (50-300 mM KCl) to distinguish specific from non-specific interactions

• SmpB mutant proteins: Generate variants with mutations in predicted RNA-binding regions to confirm interaction mechanisms

• SsrA RNA processing control: Verify that SsrA RNA processing is normal in any SmpB mutant strains by Northern blot analysis with appropriate size markers

• Ribosome association control: Include ribosome-binding assays with and without SmpB to confirm its role in SsrA-ribosome interactions

These controls help distinguish true SmpB-SsrA interactions from experimental artifacts and provide mechanistic insights into the nature of these interactions.

How can researchers address issues with recombinant P. profundum SmpB solubility and activity?

Researchers encountering solubility or activity issues with recombinant P. profundum SmpB should consider:

• Buffer optimization:

  • Test buffers with varying pH values (7.0-8.5)

  • Include stabilizing agents (glycerol 5-20%, reducing agents like DTT or β-mercaptoethanol)

  • Add low concentrations of non-ionic detergents (0.01-0.05% Triton X-100 or NP-40)

• Expression conditions:

  • Lower induction temperature (16-18°C)

  • Reduce IPTG concentration

  • Co-express with chaperones

  • Consider fusion tags that enhance solubility (MBP, SUMO)

• Purification strategy:

  • Implement gentle elution conditions

  • Include stabilizing agents throughout purification

  • Consider on-column refolding if inclusion bodies form

• Activity restoration:

  • Test activity in the presence of SsrA RNA as natural binding partner

  • Include physiologically relevant salt concentrations (200 mM KCl)

  • Consider pressure conditions that mimic natural P. profundum environment

A systematic approach testing these variables will help identify optimal conditions for maintaining solubility and activity of this deep-sea bacterial protein.

What could explain discrepancies between in vitro and in vivo results with P. profundum SmpB?

When researchers encounter discrepancies between in vitro binding/activity assays and in vivo functional studies with P. profundum SmpB, several factors may explain these differences:

• Environmental adaptations: P. profundum is a piezophilic bacterium adapted to high-pressure deep-sea environments; standard laboratory conditions may not reflect its natural operating environment

• Post-translational modifications: Potential modifications present in vivo but absent in recombinant protein

• Co-factors requirement: Additional cellular factors may be required for full SmpB functionality, as suggested by studies questioning whether macromolecules besides SmpB are necessary for SsrA function

• RNA modifications: In vitro transcribed SsrA lacks the base modifications that occur in vivo, which might affect binding characteristics

• Protein concentration effects: The in vivo concentration of SmpB (estimated to be in the micromolar range for bacterial SmpB-SsrA systems) may differ significantly from in vitro experimental conditions

To reconcile such discrepancies, researchers should consider employing techniques that bridge the gap between in vitro and in vivo conditions, such as cell extracts, semi-permeabilized cells, or reconstituted systems with additional cellular components.

How does P. profundum SmpB compare structurally and functionally to SmpB proteins from other bacterial species?

P. profundum SmpB shares the core functional characteristics of other bacterial SmpB proteins but may possess unique adaptations related to its deep-sea environment. Comparative analysis should consider:

Phylogenetic analysis across bacterial species demonstrates that SmpB is highly conserved, reflecting its essential role in the trans-translation quality control system . The observed conservation suggests that the fundamental mechanism of SmpB function is preserved across bacterial species, even as specific adaptations may occur in extremophiles like P. profundum.

How do the roles of SmpB differ in normal growth versus stress conditions in P. profundum?

While specific data on P. profundum SmpB function under different conditions is limited in the provided search results, we can infer likely differences based on known SmpB functions in other bacteria:

Evidence from other bacteria indicates that SmpB-SsrA systems become particularly important under stress conditions that increase the frequency of stalled translation, suggesting that P. profundum SmpB likely plays an enhanced role during environmental challenges, particularly those related to deep-sea pressure fluctuations .