Recombinant Chloroflexus aurantiacus Translation initiation factor IF-2 (infB), partial

How does the structure of IF-2 contribute to its thermostability in C. aurantiacus?

The thermostability of IF-2 in C. aurantiacus likely stems from specific structural adaptations that enable function at elevated temperatures. Based on studies of other proteins from this thermophilic bacterium, these adaptations typically include:

• Increased prevalence of alpha-helical structures, as observed in the related IF-3 protein which shows predominantly alpha (α) helices in its secondary structure

• Enhanced hydrophobic core packing

• Increased number of salt bridges and ionic interactions

• Reduced flexibility in loop regions

These structural elements collectively contribute to protein stability at the elevated temperatures (50-60°C) at which C. aurantiacus thrives . The tertiary structure is likely to exhibit high consistency and stability, similar to what has been observed with the IF-3 protein from this organism.

What are the physicochemical properties of C. aurantiacus translation initiation factors?

Studies on translation initiation factors from C. aurantiacus, particularly IF-3, have revealed important physicochemical properties that likely apply to IF-2 as well:

In silico investigations of C. aurantiacus translation factors have documented these properties, which are essential for function in the thermophilic environment where this organism thrives .

What are the optimal expression strategies for recombinant C. aurantiacus IF-2?

Successful expression of recombinant C. aurantiacus IF-2 requires careful optimization of multiple parameters. Based on studies with other proteins from this organism, the following approach is recommended:

• Vector selection: pET28a with N-terminal His6-tag has proven effective for C. aurantiacus proteins

• Host strain: E. coli BL21(DE3) typically provides good expression levels for thermophilic proteins

• Gene amplification: PCR from C. aurantiacus J-10-fl genomic DNA using high-fidelity polymerase

• Cloning strategy: Insertion into appropriate restriction sites (BamHI/NotI or NdeI/XhoI) of the expression vector

• Expression conditions:

  • Induction at OD600 of 0.6-0.8

  • IPTG concentration of 0.1-0.5 mM

  • Expression temperature of 30°C (compromise between host viability and proper folding)

  • Expression duration of 4-6 hours

This methodology has been successfully applied to other recombinant proteins from C. aurantiacus and would be appropriate for IF-2 expression .

How can researchers verify the functional integrity of recombinantly expressed C. aurantiacus IF-2?

Verifying the functional integrity of recombinantly expressed C. aurantiacus IF-2 requires multiple complementary approaches:

• Biochemical assays:

  • GTP binding and hydrolysis activity measurement

  • Interaction with 30S ribosomal subunits (using techniques like surface plasmon resonance)

  • Formation of initiation complexes with initiator tRNA

• Structural validation:

  • Circular dichroism to confirm secondary structure content

  • Thermal denaturation profiling to verify thermostability

  • Limited proteolysis to assess proper folding

• Complementation studies:

  • Testing whether C. aurantiacus IF-2 can functionally replace IF-2 in other bacterial systems, similar to the approach demonstrated with mammalian mitochondrial IF2 in E. coli

  • Generation of an E. coli strain with a knockout or conditional mutation of the native infB gene

  • Testing growth restoration upon expression of C. aurantiacus IF-2

• Mass spectrometry verification:

  • HPLC-ESI-MS/MS analysis to confirm protein identity and integrity

Functional verification is critical to ensure that the recombinant protein accurately represents the native C. aurantiacus IF-2 properties.

What purification challenges are specific to thermophilic translation factors, and how can they be addressed?

Purification of thermophilic translation factors like C. aurantiacus IF-2 presents unique challenges that require specific strategies:

These approaches have been successful with other thermophilic proteins and can be adapted specifically for C. aurantiacus translation factors to ensure the isolation of functionally active protein.

How can researchers analyze protein-protein interactions specific to C. aurantiacus IF-2?

Analysis of protein-protein interactions for C. aurantiacus IF-2 requires specialized approaches that account for the thermophilic nature of this protein:

• In silico interaction prediction:

  • Using the STRING program as demonstrated with IF-3 to predict interactions with the 30S ribosomal subunit and other components of the translation machinery

  • Comparative analysis with interaction networks from related organisms

• Experimental verification:

  • Pull-down assays using His-tagged IF-2 at temperatures relevant to C. aurantiacus physiology

  • Surface plasmon resonance (SPR) at elevated temperatures to measure binding kinetics

  • Microscale thermophoresis for quantitative interaction analysis

  • Crosslinking mass spectrometry to identify interaction interfaces

• Functional validation:

  • Reconstitution of initiation complexes using purified components

  • Mutagenesis of predicted interaction surfaces followed by binding studies

Protein interactions analysis has demonstrated high credence that translation factors from C. aurantiacus interact with the 30S ribosomal subunit involved in protein synthesis , providing a foundation for more detailed studies of IF-2 interactions.

What experimental approaches can determine the rate-limiting steps in translation initiation mediated by C. aurantiacus IF-2?

Determining rate-limiting steps in translation initiation mediated by C. aurantiacus IF-2 requires sophisticated kinetic analyses:

• Pre-steady state kinetic measurements:

  • Stopped-flow fluorescence spectroscopy to monitor conformational changes during initiation

  • Rapid quench-flow techniques to capture transient intermediates

  • Single-molecule FRET to observe individual initiation events in real-time

• Component variation experiments:

  • Systematic variation of initiation component concentrations to identify rate-determining steps

  • Temperature-dependent kinetic studies to determine activation energies of individual steps

  • Competition experiments between native and modified components

• Comparative analysis:

  • Parallel kinetic studies with IF-2 from mesophilic organisms to identify thermophile-specific kinetic parameters

  • Analysis of how temperature affects rate-limiting steps differently in thermophilic versus mesophilic systems

These approaches would provide insights into how C. aurantiacus IF-2 has adapted its kinetic properties to function optimally at elevated temperatures while maintaining the essential function of facilitating translation initiation.

How does biotinylation impact the function of translation factors in C. aurantiacus?

While direct studies on biotinylation of C. aurantiacus IF-2 are not available, research on other C. aurantiacus proteins provides relevant insights:

• Biotinylation mechanisms:

  • C. aurantiacus possesses its own BirA enzyme (Caur_0481) capable of biotinylating proteins with appropriate recognition sequences

  • Heterologous biotinylation is possible, as demonstrated by successful biotinylation of C. aurantiacus proteins by E. coli BirA

• Functional significance:

  • Biotinylation can promote protein-protein interactions, as shown for other C. aurantiacus proteins where biotinylation of a BCCP domain enhanced interactions between protein subunits

  • This post-translational modification may play a role in regulating the assembly of multi-component complexes

• Experimental considerations:

  • When expressing recombinant C. aurantiacus IF-2, co-expression with BirA might be necessary if the protein contains biotinylation sites

  • Biotinylation status should be verified as part of protein characterization

Although translation factors are not typically biotinylated proteins, this post-translational modification mechanism in C. aurantiacus illustrates the sophisticated regulatory systems that may influence protein function in this organism.

How does C. aurantiacus IF-2 compare structurally and functionally to homologs in other thermophilic bacteria?

Comparative analysis of C. aurantiacus IF-2 with homologs from other thermophilic bacteria reveals important insights into convergent and divergent evolutionary adaptations:

Phylogenetic analyses of other C. aurantiacus proteins have indicated that they originated from distinct Chloroflexi species , suggesting that IF-2 would likely show similar evolutionary patterns specific to this phylum while maintaining the core features required for translation initiation.

Can C. aurantiacus IF-2 complement deficiencies in translation initiation systems of other organisms?

The potential for functional complementation between C. aurantiacus IF-2 and other translation systems can be assessed based on complementation studies with other translation factors:

• Evidence from related systems:

  • Mammalian mitochondrial IF2 has been shown to functionally replace E. coli IF2 in a knockout strain (ΔinfB::KanR)

  • This suggests that despite sequence divergence and adaptation to different environments, core functional features of IF-2 are conserved

• Experimental approach:

  • Generation of an E. coli strain with the infB gene replaced by a kanamycin resistance marker (ΔinfB::KanR)

  • Introduction of a plasmid expressing C. aurantiacus IF-2

  • Verification of genomic replacement using diagnostic PCR as demonstrated with other systems

  • Assessment of growth and protein synthesis capacity

• Factors affecting complementation:

  • Temperature compatibility (thermophilic IF-2 functioning at mesophilic temperatures)

  • Interaction compatibility with other components of the host translation machinery

  • Potential requirements for co-expression of other C. aurantiacus factors

Successful complementation would provide strong evidence for the conservation of fundamental mechanisms in translation initiation across diverse bacterial species despite adaptation to different thermal environments.

What evolutionary insights can be gained from studying the infB gene in C. aurantiacus?

Studying the infB gene in C. aurantiacus offers valuable evolutionary insights:

• Adaptation to thermophily:

  • Analysis of codon usage and amino acid composition can reveal thermophilic adaptations at both nucleotide and protein levels

  • Comparison with mesophilic homologs can identify specific substitutions associated with thermal adaptation

• Evolutionary history of Chloroflexi:

  • As a member of the ancient Chloroflexi phylum, C. aurantiacus translation factors may preserve features of early bacterial translation systems

  • C. aurantiacus employs a unique 3-hydroxypropionate bi-cycle rather than the Calvin cycle for carbon fixation , suggesting unique evolutionary history

• Horizontal gene transfer assessment:

  • Phylogenetic analysis can reveal whether infB evolved vertically within Chloroflexi or was acquired horizontally

  • Analysis of GC content and codon bias can identify potential foreign origin

• Molecular clock applications:

  • As highly conserved proteins, translation factors can serve as molecular clocks for dating evolutionary divergence

  • Rate of sequence evolution in thermophiles may differ from mesophiles, providing insights into temperature effects on molecular evolution

These evolutionary analyses contribute to our understanding of both the specific adaptations of C. aurantiacus and broader patterns of translation system evolution across the bacterial domain.

How can site-directed mutagenesis of C. aurantiacus IF-2 reveal functional domains critical for thermostability?

Site-directed mutagenesis offers a powerful approach to identify specific residues and domains in C. aurantiacus IF-2 that contribute to thermostability:

• Target selection strategy:

  • Compare sequences with mesophilic homologs to identify thermophile-specific residues

  • Focus on charged residues that may form salt bridges

  • Target hydrophobic core residues that might contribute to stabilization

  • Examine loops and regions with different conformational flexibility

• Experimental design:

  • Create single-point mutations replacing thermophile-specific residues with mesophilic equivalents

  • Generate domain-swapping variants between thermophilic and mesophilic IF-2

  • Create progressive truncations to identify stabilizing domains

• Assessment methods:

  • Thermal stability assays (differential scanning calorimetry, thermal shift assays)

  • Activity measurements at various temperatures

  • Structural analysis of mutants (CD spectroscopy, limited proteolysis)

  • Complementation ability in infB knockout strains

• Data interpretation framework:

  • Classify mutations based on their effects (destabilizing, neutral, or enhancing)

  • Map critical residues to the three-dimensional structure

  • Identify cooperative networks of stabilizing interactions

This systematic approach would generate a detailed map of the structural features that enable C. aurantiacus IF-2 to function at elevated temperatures while maintaining the precision required for accurate translation initiation.

What high-resolution structural techniques are most appropriate for studying C. aurantiacus translation factors?

Multiple complementary structural techniques are needed to fully characterize C. aurantiacus translation factors:

The tertiary-structure model developed through these approaches would be expected to exhibit reasonably high consistency based on various quality assessment methods, similar to what has been observed for other C. aurantiacus proteins . Integration of these techniques would provide comprehensive insights into how the structure of C. aurantiacus IF-2 relates to its function in protein synthesis at elevated temperatures.

How can in silico approaches contribute to understanding C. aurantiacus translation factor function?

In silico approaches provide valuable insights into C. aurantiacus translation factor function, complementing experimental studies:

• Structural prediction and analysis:

  • Homology modeling based on related structures

  • Ab initio structure prediction for unique domains

  • Molecular dynamics simulations at elevated temperatures to assess thermostability

  • Prediction of secondary structure elements, showing the prevalence of alpha-helical structures

• Functional annotation:

  • Gene ontology assignment

  • Functional domain prediction

  • Binding site identification

  • Catalytic residue prediction

• Interaction network analysis:

  • Prediction of protein-protein interactions using tools like STRING

  • Identification of interaction partners within the translation machinery

  • Comparative analysis with interaction networks from related organisms

• Evolutionary analysis:

  • Multiple sequence alignment with homologs

  • Identification of conserved residues across thermophiles

  • Phylogenetic reconstruction to trace evolutionary history

  • Detection of signatures of selection

These in silico approaches have been successfully applied to other C. aurantiacus proteins, revealing their physicochemical properties, subcellular location, three-dimensional structure, and functional characteristics , providing a foundation for understanding IF-2 and other translation factors from this organism.

What experimental controls are essential when studying recombinant C. aurantiacus translation factors?

Rigorous experimental design for studies of recombinant C. aurantiacus translation factors requires several essential controls:

• Expression and purification controls:

  • Empty vector control to identify host-derived contaminants

  • Purification of a known thermostable protein using identical protocols

  • Non-biotinylated controls when studying biotinylation effects

  • Mass spectrometry verification of protein identity and integrity

• Functional assay controls:

  • Heat-inactivated protein to distinguish specific activity from artifacts

  • Concentration-matched BSA or other non-relevant proteins

  • Homologous proteins from mesophilic organisms for comparative analysis

  • Testing at both physiological (50-60°C) and standard (37°C) temperatures

• Complementation study controls:

  • Wild-type strain verification using diagnostic PCR

  • Controls for physically unlinked genes to verify DNA quality

  • Positive control using known complementing genes (e.g., E. coli IF2 for infB knockout)

• Structural analysis controls:

  • Analysis of proteins with known structure using identical methods

  • Multiple quality assessment methods to verify structural models

These controls ensure that experimental observations accurately reflect the properties of C. aurantiacus translation factors rather than artifacts or experimental variables.

How should researchers design experiments to distinguish temperature-specific adaptations from other functional features in C. aurantiacus IF-2?

Distinguishing temperature-specific adaptations from other functional features requires carefully designed comparative experiments:

• Temperature-dependent functional assays:

  • Activity measurements across a temperature range (20-70°C)

  • Determination of temperature optima and Arrhenius plots

  • Comparison with mesophilic homologs under identical conditions

  • Assessment of thermal inactivation rates

• Chimeric protein analysis:

  • Creation of hybrid proteins with domains from thermophilic and mesophilic IF-2

  • Systematic domain swapping to isolate thermostable regions

  • Functional testing of chimeras at various temperatures

• Comparative mutagenesis:

  • Introduction of "thermophilic" residues into mesophilic IF-2

  • Introduction of "mesophilic" residues into C. aurantiacus IF-2

  • Testing whether thermal properties change independently of other functional parameters

• Structural analysis under variable conditions:

  • Structural characterization at different temperatures

  • Identification of temperature-dependent conformational changes

  • Comparison with temperature effects on mesophilic homologs

This multifaceted approach separates features that specifically contribute to thermostability from those that are essential for IF-2 function across all bacterial species, providing insights into the molecular basis of thermal adaptation.

What are the critical considerations for storage and handling of recombinant C. aurantiacus proteins to maintain their integrity?

Proper storage and handling of recombinant C. aurantiacus proteins requires protocols that preserve their unique properties:

These practices leverage the inherent stability of thermophilic proteins while protecting against specific modes of degradation, ensuring that experimental results accurately reflect the native properties of C. aurantiacus translation factors.