Recombinant Shewanella baltica Translation initiation factor IF-2 (infB), partial

What is Shewanella baltica and why is it significant in research?

Shewanella baltica is a psychrotropic (cold-tolerant) bacterium that has been identified as the main H2S-producing organism in iced marine fish from the Baltic Sea. Previously misidentified as S. putrefaciens, it dominates the H2S-producing bacterial population during cold storage of marine fish . S. baltica can utilize citrate and sucrose as sole carbon sources, typically cannot grow at 37°C, and has a G+C mol% content of 46-47% . This organism grows well in cold conditions, including at 0°C in cod juice , making it an excellent model for studying bacterial adaptation to low temperatures and protein function in psychrotropic organisms.

What is Translation initiation factor IF-2 (infB) and what role does it play in bacterial protein synthesis?

Translation initiation factor IF-2 is a critical protein involved in the initiation phase of protein synthesis. This GTP-binding protein facilitates the binding of initiator tRNA (fMet-tRNA) to the 30S ribosomal subunit during formation of the translation initiation complex. In bacteria like Shewanella baltica, IF-2 is particularly important for maintaining protein synthesis under stress conditions such as cold temperatures. The protein ensures accurate translation by promoting correct positioning of the start codon and initiator tRNA on the ribosome, a process that may require special adaptations in cold-tolerant organisms.

Why study the partial recombinant IF-2 from Shewanella baltica specifically?

Studying partial recombinant IF-2 from S. baltica provides valuable insights into how psychrotropic bacteria maintain protein synthesis machinery at low temperatures. The partial protein (as indicated in product information) likely contains key domains responsible for cold adaptation while being easier to express and purify than the full-length protein. Comparative studies between IF-2 from S. baltica and mesophilic bacteria can reveal structural and functional adaptations that enable efficient translation initiation at low temperatures, contributing to our understanding of bacterial adaptation mechanisms and potentially identifying features that could be applied in biotechnology.

How might the structure of S. baltica IF-2 differ from IF-2 in mesophilic bacteria to enable function at low temperatures?

S. baltica IF-2 likely incorporates structural adaptations that optimize function in cold environments. These adaptations may include: increased flexibility in loop regions to maintain activity despite reduced molecular kinetic energy at low temperatures; altered amino acid composition with fewer proline residues and potentially more glycine to increase backbone flexibility; modified surface charge distribution to maintain proper protein-protein interactions at low temperatures; and adjustments in the GTP-binding domain to ensure proper nucleotide binding and hydrolysis kinetics in cold conditions. Detailed structural studies comparing IF-2 from S. baltica with mesophilic homologs would reveal specific cold-adaptive features that contribute to the psychrotropic lifestyle of this organism.

What contradictions exist in current research regarding cold adaptation of translation machinery?

Current research on cold adaptation of translation machinery presents several contradictions. While increased structural flexibility is generally associated with cold adaptation, excessive flexibility can compromise thermal stability. The specific regions requiring flexibility versus stability in translation factors remain debated. Additionally, some studies suggest cold adaptation occurs primarily through changes in protein sequence, while others emphasize regulation of expression levels. There are conflicting reports about whether cold adaptation of translation initiation factors involves modifications to core catalytic domains or primarily to peripheral regions. These contradictions highlight the need for comprehensive studies of proteins like S. baltica IF-2 to establish clearer principles of translation adaptation to cold environments.

How do post-translational modifications potentially regulate S. baltica IF-2 function during temperature shifts?

Post-translational modifications likely play crucial but understudied roles in regulating S. baltica IF-2 during temperature shifts. Phosphorylation at specific residues might change in response to temperature fluctuations, affecting GTPase activity or interactions with ribosomes. Other potential modifications include methylation, acetylation, or even specific proteolytic processing that could generate functional variants optimized for different temperatures. These modifications might create a more responsive translation system capable of maintaining activity across the temperature range S. baltica encounters in marine environments (0-25°C). Comprehensive proteomic analysis of IF-2 isolated from cells grown at different temperatures would provide valuable insights into these regulatory mechanisms.

What are the optimal conditions for expressing and purifying recombinant S. baltica IF-2?

For optimal expression and purification of recombinant S. baltica IF-2, several specific conditions should be considered. Expression in E. coli is effective , but should be performed at lower temperatures (15-20°C rather than 37°C) to improve folding of this psychrotropic protein. Induction with low IPTG concentrations (0.1-0.5 mM) for extended periods (overnight) typically yields better results for cold-adapted proteins. Purification should employ immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography to achieve >85% purity as verified by SDS-PAGE . The purified protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, and 5-50% glycerol should be added for long-term storage . Storage at -20°C is suitable for short periods, but -80°C is preferable for extended storage to maintain stability .

What considerations are important when designing functional assays for S. baltica IF-2 at different temperatures?

When designing functional assays for S. baltica IF-2 across temperature ranges, several factors require careful consideration. Buffer components must be checked for temperature-dependent pH shifts using temperature-compensated pH meters, as many buffers exhibit significant pH changes at low temperatures. Reaction kinetics will vary dramatically with temperature, requiring adjusted incubation times (longer at lower temperatures). All components (ribosomes, tRNAs, mRNAs) should be pre-equilibrated to test temperatures for accurate results. Control reactions using mesophilic IF-2 variants should be included at each temperature point for meaningful comparisons. For GTPase assays, GTP stability must be verified at all test temperatures. Specialized equipment for maintaining precise low temperatures (0-4°C) during experiments is essential, with continuous temperature monitoring to ensure stability throughout extended assays.

How should researchers interpret contradictory findings regarding S. baltica IF-2 activity at different temperatures?

When faced with contradictory findings regarding S. baltica IF-2 temperature-dependent activity, researchers should systematically evaluate several factors. Experimental conditions (buffer composition, pH, salt concentration) can significantly affect results and must be meticulously standardized across studies. The partial nature of the recombinant protein may affect activity measurements if different constructs contain different functional domains. Direct translation initiation assays versus indirect GTPase activity measurements may yield apparently contradictory results but actually reflect different aspects of IF-2 function. Interactions with different components (ribosomes from different sources, varied mRNA substrates) can influence activity. To resolve contradictions, researchers should employ multiple independent methodologies and collaborate with other groups using identical protein preparations and protocols.

How can researchers distinguish between specific cold adaptations in S. baltica IF-2 versus general features of translation initiation factors?

To distinguish between specific cold adaptations and general features, researchers must employ controlled comparative analyses. Phylogenetic analysis should include IF-2 proteins from multiple Shewanella species with diverse temperature preferences but similar evolutionary distances. Domain-specific analysis can identify if cold adaptations cluster in particular functional regions of the protein. Site-directed mutagenesis of predicted adaptive sites followed by functional testing at different temperatures (0-37°C) can confirm their significance. Heterologous expression of S. baltica IF-2 in mesophilic hosts with subsequent growth tests at low temperatures can reveal if the protein alone confers cold-adaptive advantages. The partial nature of the recombinant protein should be considered, as some adaptive features may reside in regions not included in the construct.

How might insights from S. baltica IF-2 research contribute to biotechnological applications?

Research on S. baltica IF-2 could enable several biotechnological innovations. Understanding cold-adapted protein synthesis machinery could lead to improved cell-free protein synthesis systems functioning efficiently at lower temperatures (4-15°C), reducing energy costs and preserving heat-sensitive products. Cold-adaptive features identified in IF-2 could be engineered into expression systems for recombinant protein production, potentially improving folding of difficult proteins at lower temperatures. Additionally, principles learned from studying how S. baltica maintains efficient translation at low temperatures could inform strategies for enhancing crop cold tolerance through modification of translation machinery or developing biopharmaceuticals with extended storage stability at refrigerated temperatures. The reconstitution and storage protocols developed for the recombinant protein also provide valuable information for handling cold-adapted proteins in biotechnological applications.

What are the most promising techniques for studying interactions between S. baltica IF-2 and other components of the translation machinery?

Several advanced techniques are particularly promising for studying IF-2 interactions. Cryo-electron microscopy can capture IF-2 bound to ribosomes under near-native conditions at different temperatures, revealing how binding dynamics change with temperature. Surface plasmon resonance and microscale thermophoresis can quantitatively measure binding kinetics between IF-2 and its partners (ribosomes, initiator tRNA, GTP) across temperature ranges. Hydrogen-deuterium exchange mass spectrometry can identify interface regions between IF-2 and binding partners that may be modified for cold adaptation. For in vivo studies, fluorescence resonance energy transfer (FRET) systems with fluorescently labeled IF-2 and ribosomal components can monitor interactions in living cells at different temperatures. When using partial recombinant IF-2 , researchers must consider whether all interaction surfaces are present in the construct.