Recombinant Desulfotalea psychrophila Elongation factor P (efp)

What is Elongation Factor P (EFP) and what specific role does it play in Desulfotalea psychrophila?

Elongation Factor P (EFP) is a universally conserved bacterial translation factor that alleviates ribosome stalling during the synthesis of proteins containing consecutive proline residues. In Desulfotalea psychrophila, a sulfate-reducing delta-proteobacterium that thrives in permanently cold Arctic sediments below 0°C, EFP likely plays a critical role in maintaining efficient protein synthesis under extreme cold conditions .
The D. psychrophila genome (3,523,383 bp circular chromosome with 3,118 predicted genes) encodes this essential translation factor that structurally resembles tRNA and binds between the P and E sites of the ribosome. Unlike other elongation factors that are dynamically associated with the ribosome during protein synthesis, EFP is specifically recruited to overcome translational challenges posed by polyproline motifs.
Methodological approach for studying D. psychrophila EFP function:

• Genomic identification through comparative analysis with known efp genes

• Recombinant expression at low temperatures (10-15°C) to maintain native structure

• In vitro translation assays at various temperatures (0-20°C) using polyproline reporters

• Complementation studies in efp-deficient E. coli strains

How does the sequence and structure of D. psychrophila EFP differ from mesophilic bacterial homologs?

D. psychrophila EFP exhibits characteristic adaptations typical of cold-active proteins when compared to its mesophilic counterparts. While specific structural data for D. psychrophila EFP is not provided in the search results, comparative analysis with other psychrophilic proteins suggests these likely adaptations:

• Multiple sequence alignment comparing D. psychrophila EFP with homologs from various thermal classes

• Homology modeling based on known EFP structures

• Molecular dynamics simulations at various temperatures

• Circular dichroism spectroscopy to assess secondary structure stability

What expression systems are optimal for producing functional recombinant D. psychrophila EFP?

Several expression systems can be employed for recombinant D. psychrophila EFP production, each with specific advantages:

• Incorporate solubility-enhancing tags (MBP, SUMO, or TrxA) to improve folding

• Use enriched media supplemented with 5% glycerol to prevent cold stress

• Maintain strict temperature control during induction and harvesting

• Validate protein activity immediately after purification at low temperatures

What purification strategies yield active D. psychrophila EFP with highest purity?

Multi-step purification approach optimized for psychrophilic proteins:

• Initial capture:

  • Immobilized metal affinity chromatography (IMAC) using Ni-NTA at 4°C

  • Buffer: 50mM Tris-HCl pH 8.0, 300mM NaCl, 10% glycerol, 5mM β-mercaptoethanol

• Intermediate purification:

  • Ion exchange chromatography (typically Q-Sepharose at pH 8.0)

  • Size exclusion chromatography using Superdex 75

• Activity validation:

  • In vitro translation assay using polyproline reporter peptides

  • Thermal shift assay to confirm proper folding
    Methodological consideration for low-temperature proteins:

• All purification steps should be performed at 4°C or lower

• Include cryoprotectants (10% glycerol) in all buffers to prevent cold denaturation

• Use gentle elution gradients to maintain structural integrity

• Validate final product with mass spectrometry to confirm post-translational modifications

How can researchers confirm the structural integrity of purified recombinant D. psychrophila EFP?

Multiple complementary techniques should be employed to verify structural integrity:

• Translation activity assay using a polyproline reporter system

• Ribosome binding assays at temperatures ranging from 0-20°C

• Comparison with recombinant EFP from mesophilic organisms

How does temperature affect the kinetics of D. psychrophila EFP in translation systems?

Temperature profoundly impacts the kinetic parameters of D. psychrophila EFP, with important implications for protein synthesis in cold environments:

• Stopped-flow fluorescence spectroscopy to measure binding kinetics

• In vitro translation assays at controlled temperatures

• Ribosome binding assays with purified components

• Real-time monitoring of polyproline peptide synthesis
  The psychrophilic adaptation of D. psychrophila EFP likely involves:

• Lower activation energy (Ea) for catalysis

• Optimized binding kinetics at low temperatures

• Potentially different rate-limiting steps compared to mesophilic homologs

• Temperature-dependent conformational dynamics

What post-translational modifications are essential for D. psychrophila EFP function?

In many bacteria, EFP requires specific post-translational modifications (PTMs) for full activity, particularly for alleviating ribosome stalling at polyproline sequences. The D. psychrophila genome analysis suggests potential modification pathways:

• LC-MS/MS analysis of native D. psychrophila EFP

• In vitro reconstitution of modification reactions at various temperatures

• Mutagenesis of target residues to assess impact on function

• Co-expression with modification enzymes in recombinant systems
  The cold adaptation of D. psychrophila may include unique modifications or altered modification efficiency, which would have implications for proper recombinant production.

How does D. psychrophila EFP interact with ribosomes to facilitate polyproline translation at low temperatures?

D. psychrophila EFP likely exhibits specialized interactions with the translation machinery to maintain efficient protein synthesis in cold environments:

• Cryo-EM structures of D. psychrophila EFP bound to ribosomes at 4°C

• Comparative binding studies with mesophilic components

• Site-directed mutagenesis of interaction interfaces

• Kinetic analysis of stalled ribosome rescue
  As shown in research with other translation factors, D. psychrophila EFP likely works in concert with additional cold-adapted components of the translation machinery, including potential coordination with elongation factors mentioned in the search results .

What structural features of D. psychrophila EFP contribute to its cold adaptation?

While specific structural data for D. psychrophila EFP is not available in the search results, comparative analysis with other cold-adapted proteins suggests several probable features:

• X-ray crystallography at multiple temperatures

• Hydrogen-deuterium exchange mass spectrometry to map flexibility

• Molecular dynamics simulations at low temperatures

• Site-directed mutagenesis of predicted flexibility-enhancing residues
  The D. psychrophila genome (3,523,383 bp) encodes proteins adapted to function in permanently cold Arctic sediments, and EFP would be expected to share common cold-adaptation strategies with other proteins from this organism .

How can D. psychrophila EFP be engineered to optimize its function for biotechnological applications?

Strategic engineering of D. psychrophila EFP could enhance its utility in various applications:

• Rational design based on structural comparison with mesophilic EFPs

• Directed evolution at various temperatures

• Domain swapping with EFPs from different temperature classes

• High-throughput screening using polyproline translation reporters
  Applications in biotechnology could include:

• Enhancement of cold-active expression systems

• Improved cell-free protein synthesis at low temperatures

• Development of biosensors for environmental monitoring

How does D. psychrophila EFP contribute to global gene expression patterns at low temperatures?

D. psychrophila EFP likely plays a regulatory role in shaping the psychrophilic proteome through preferential translation of specific mRNAs:

• Genome-wide analysis of polyproline motif distribution in D. psychrophila

• Ribosome profiling at different temperatures (0°C, 4°C, 10°C)

• Proteomics comparison of wild-type and EFP-depleted cells

• RNA-seq to identify transcripts with high ribosome occupancy at proline codons
  Research by Clark and Fields (mentioned in the search results) suggests that D. psychrophila's growth characteristics and biofilm formation involve coordinated regulation of gene expression during adaptation to different conditions . EFP likely contributes to these adaptation processes by ensuring efficient translation of key proteins containing polyproline motifs.

How does D. psychrophila EFP compare functionally with EFPs from other psychrophilic bacteria?

Comparative analysis of EFPs from different psychrophilic bacteria reveals evolutionary strategies for cold adaptation:

• Comparative genomics of efp genes and modification enzymes

• Heterologous complementation studies in efp-deficient strains

• In vitro activity assays under identical conditions

• Structural comparison through homology modeling and crystallography
  This comparison provides insights into convergent and divergent evolutionary strategies for adaptation to cold environments. The D. psychrophila genome contains adaptations for its specific ecological niche in permanently cold marine sediments where it contributes to global carbon and sulfur cycles .

What is the role of D. psychrophila EFP in translating cold-essential proteins during temperature stress?

During temperature fluctuations, D. psychrophila EFP likely plays a critical role in maintaining translation of essential proteins:

• Temperature shift experiments with transcriptome and proteome analysis

• Identification of polyproline-containing proteins induced during stress

• Pulse-chase labeling to measure translation rates during temperature transitions

• EFP depletion studies to assess sensitivity to temperature fluctuations
  The transcriptomic analysis of Desulfovibrio vulgaris (a related sulfate-reducing bacterium) during stress conditions revealed complex gene expression changes , and similar mechanisms likely operate in D. psychrophila, with EFP playing a crucial role in translating key stress response proteins.

How can recombinant D. psychrophila EFP improve cold-adapted expression systems for difficult proteins?

The unique properties of D. psychrophila EFP can be leveraged to enhance protein expression at low temperatures:

• Co-expression of D. psychrophila EFP with target proteins

• Development of cold-adapted cell-free translation systems

• Engineering of E. coli strains with psychrophilic translation machinery

• Temperature-controlled bioreactors with optimized expression parameters
  Experimental data should be collected on:

• Expression yields at different temperatures (4°C, 10°C, 15°C, 20°C)

• Solubility comparison with conventional systems

• Activity of proteins expressed with and without D. psychrophila EFP

• Cost-benefit analysis for industrial applications

How do post-translational modifications of D. psychrophila EFP differ in their temperature dependence compared to mesophilic modifications?

The enzymes responsible for EFP modifications in D. psychrophila likely show cold adaptation, affecting modification efficiency at different temperatures:

• Recombinant expression of modification enzymes

• In vitro modification assays at temperatures from 0-37°C

• Mass spectrometry to quantify modification efficiency

• Structural studies of enzyme-substrate complexes
  The modifications of D. psychrophila EFP likely show more efficient installation at low temperatures compared to their mesophilic counterparts, which would represent an important adaptation for ensuring translation efficiency in permanently cold environments where D. psychrophila thrives .