Recombinant Ignicoccus hospitalis Signal recognition particle 19 kDa protein (srp19)

What is the biological role of the srp19 protein in Ignicoccus hospitalis?

The srp19 protein in I. hospitalis is a critical component of the Signal Recognition Particle complex, which facilitates protein targeting to cellular membranes. In I. hospitalis, this function is particularly significant due to the organism's unique cellular architecture featuring an outer cellular membrane (OCM) and inner membrane (IM) enclosing an intermembrane compartment (IMC). The srp19 protein likely plays a key role in directing proteins to these distinct membrane systems, particularly those involved in the endomembrane system with secretory functions . The protein assists in binding signal recognition particle RNA and maintaining the structural integrity of the SRP complex required for proper protein translocation.

What expression systems are most effective for producing recombinant I. hospitalis srp19?

For recombinant expression of I. hospitalis srp19, E. coli-based expression systems using codon-optimized genes have proven effective. Based on protocols used for other I. hospitalis proteins, the gene can be synthesized with codon optimization, cloned into expression vectors like pJExpress, and expressed in E. coli with a C-terminal 6xHis tag for purification . When establishing your expression system, consider the following protocol steps:

• Codon optimization for E. coli expression

• Gene synthesis and cloning into an appropriate expression vector

• Expression in E. coli under optimized conditions

• Purification using Ni-resin affinity chromatography

• Verification of protein integrity by SDS-PAGE and Western blotting

This approach has been successfully used for other I. hospitalis proteins and can be adapted for srp19 expression.

How can I verify the structural integrity of purified recombinant I. hospitalis srp19?

Verification of recombinant I. hospitalis srp19 structural integrity requires a multi-method approach:

• Circular Dichroism (CD) Spectroscopy: To assess secondary structure elements and proper folding

• Size Exclusion Chromatography: To confirm the monomeric state and absence of aggregation

• Thermal Shift Assays: To evaluate protein stability under various buffer conditions

• RNA Binding Assays: To confirm functional activity through binding to SRP RNA

• Limited Proteolysis: To verify the compact, properly folded structure

Compare your results with known archaeal SRP19 proteins to ensure that your recombinant protein maintains the expected structural characteristics necessary for functional studies.

What challenges are associated with working with recombinant proteins from hyperthermophilic archaea like I. hospitalis?

Working with recombinant proteins from hyperthermophilic archaea presents several distinct challenges:

• Temperature Adaptation: I. hospitalis proteins are adapted to function at 90°C , while standard laboratory protocols typically operate at much lower temperatures

• Codon Usage Bias: The significant difference in codon usage between I. hospitalis and expression hosts like E. coli necessitates codon optimization

• Post-translational Modifications: Archaeal-specific modifications may be absent in bacterial expression systems

• Protein Folding: The hyperthermophilic nature of I. hospitalis proteins may lead to improper folding at lower temperatures

• Buffer Compatibility: Proteins evolved to function in high-salt, high-temperature environments may require specialized buffers to maintain stability

To address these challenges, researchers should consider expression with thermostable chaperones, testing protein functionality at elevated temperatures, and including stabilizing agents in purification buffers.

How does the structure and function of I. hospitalis srp19 compare to its homologs in other domains of life?

I. hospitalis srp19, as a member of the TACK superphylum of archaea , represents an evolutionarily significant position for studying SRP complex evolution. Comparative analysis reveals:

The archaeal srp19 from I. hospitalis likely represents an ancestral form that demonstrates how membrane-targeting systems evolved before the emergence of the complex eukaryotic endomembrane system. The unusual cellular compartmentalization of I. hospitalis, with its complex endomembrane system , makes this comparison particularly relevant for understanding the evolution of protein targeting systems.

What is the role of srp19 in the unique cellular compartmentalization of I. hospitalis?

I. hospitalis possesses a highly unusual cellular anatomy with a complex and dynamic endomembrane system consisting of cytoplasmic protrusions with secretory functions . The srp19 protein likely plays a crucial role in directing proteins to specific membranes within this complex cellular architecture.

Given that the intermembrane compartment (IMC) of I. hospitalis makes up approximately 40% of the whole cell volume , proper protein targeting is essential for maintaining this unique cellular organization. The srp19 protein may be involved in:

• Directing proteins to the outer cellular membrane (OCM)

• Facilitating protein transport to the inner membrane (IM)

• Coordinating protein delivery to the cytoplasmic protrusions

• Managing protein secretion into the IMC

This protein targeting system may be particularly important for I. hospitalis interactions with N. equitans, as contact between N. equitans cytoplasm and the I. hospitalis endomembrane system has been observed .

How does the presence of Nanoarchaeum equitans affect the expression and function of srp19 in I. hospitalis?

The close association between I. hospitalis and N. equitans likely influences the expression and function of srp19. Proteomic analyses of I. hospitalis revealed that:

• The presence of N. equitans induces significant changes in the protein expression patterns of I. hospitalis

• Gene Set Enrichment Analysis (GSEA) shows that in co-culture with N. equitans, I. hospitalis exhibits increased expression of proteins involved in membrane biogenesis and post-translational protein modification

• The presence of N. equitans appears to trigger stress responses in I. hospitalis, which may affect protein targeting systems

While specific data on srp19 regulation is not directly available, the observed changes in membrane-related proteins suggest that protein targeting systems, including the SRP complex, are likely modulated during this inter-archaeal relationship.

What experimental approaches are most effective for studying the interaction between recombinant I. hospitalis srp19 and SRP RNA?

To study interactions between recombinant I. hospitalis srp19 and SRP RNA, consider these advanced methodological approaches:

• Electrophoretic Mobility Shift Assays (EMSA): To determine binding affinities and kinetics between purified srp19 and synthesized SRP RNA

• Surface Plasmon Resonance (SPR): For real-time analysis of protein-RNA binding dynamics

• Isothermal Titration Calorimetry (ITC): To measure thermodynamic parameters of the interaction

• UV Cross-linking: To map specific contact sites between srp19 and SRP RNA

• Fluorescence Anisotropy: To measure binding under various temperature and salt conditions that mimic the natural hyperthermophilic environment

These experiments should be conducted at elevated temperatures (when possible) to mimic the natural 90°C environment where I. hospitalis thrives , potentially revealing unique binding characteristics adapted to hyperthermophilic conditions.

How can cryo-electron microscopy be optimized for structural studies of the I. hospitalis SRP complex containing srp19?

Cryo-electron microscopy (cryo-EM) studies of the I. hospitalis SRP complex require specialized approaches:

• Sample Preparation Optimization:

  • Avoid centrifugation when possible to preserve native structure, similar to protocols used for I. hospitalis cellular studies

  • Consider using high-pressure freezing techniques as used in ultrastructural studies of I. hospitalis

  • Test various buffer compositions to maintain complex stability

• Data Collection Parameters:

  • Collect data at multiple defocus values to enhance contrast

  • Use energy filters to improve signal-to-noise ratio

  • Consider tilt series collection for improved 3D reconstruction

• Processing Considerations:

  • Apply specialized classification approaches to identify heterogeneous complexes

  • Utilize focused refinement on the srp19-RNA interface

  • Implement temperature-factor sharpening optimized for archaeal complexes

This methodology builds upon approaches used for ultrastructural studies of I. hospitalis using electron tomography and focused ion beam scanning electron microscopy (FIB/SEM) .

What is the optimal strategy for generating antibodies against I. hospitalis srp19 for immunolocalization studies?

Based on successful antibody generation strategies used for other I. hospitalis proteins, the recommended approach for srp19 antibody production includes:

• Protein Preparation:

  • Express recombinant srp19 with a C-terminal 6xHis tag

  • Purify using Ni-resin affinity chromatography

  • Verify purity by SDS-PAGE

• Immunization Protocol:

  • Use purified protein to raise polyclonal antibodies in rabbits

  • Consider commercial antibody production services like Covance Research Products Inc.

  • For monoclonal antibodies, select hybridomas with high specificity for conformational epitopes

• Antibody Validation:

  • Test antibody specificity by Western blotting against whole cell lysates

  • Perform pre-adsorption controls with purified antigen

  • Validate cross-reactivity with native protein by immunoprecipitation

• Immunolocalization Protocol Optimization:

  • Use high-pressure freezing and freeze-substitution methods as applied for other I. hospitalis proteins

  • Test various fixation and permeabilization conditions

  • Optimize antibody concentration and incubation conditions

This approach has been successfully used for generating antibodies against other I. hospitalis proteins, including ATP synthase components .

How can I design experiments to investigate the temperature-dependent structural changes in I. hospitalis srp19?

To investigate temperature-dependent structural changes in I. hospitalis srp19, a comprehensive experimental design should include:

• Differential Scanning Calorimetry (DSC):

  • Measure thermal unfolding transitions from 25°C to 95°C

  • Determine transition temperatures and enthalpy changes

  • Compare stability in various buffer conditions

• Temperature-Dependent Circular Dichroism (CD):

  • Monitor secondary structure changes across temperature range

  • Identify cooperative unfolding transitions

  • Determine reversibility of thermal denaturation

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Collect 1H-15N HSQC spectra at increasing temperatures

  • Track chemical shift perturbations with temperature

  • Identify regions with differential temperature sensitivity

• Small-Angle X-ray Scattering (SAXS):

  • Measure radius of gyration changes with temperature

  • Assess conformational ensemble at different temperatures

  • Generate temperature-dependent structural models

• Functional Assays at Variable Temperatures:

  • Test RNA binding capacity from 37°C to 90°C

  • Compare activity to mesophilic homologs

  • Identify temperature optima for different functions

This multi-technique approach will provide comprehensive insights into how I. hospitalis srp19 maintains structure and function at the extreme temperatures (90°C) where this organism thrives .

What controls are essential when analyzing the impact of srp19 mutations on protein targeting in I. hospitalis?

When analyzing the impact of srp19 mutations on protein targeting in I. hospitalis, implement these essential controls:

These controls are particularly important given the complex membrane organization of I. hospitalis, which includes distinct inner and outer membranes enclosing a voluminous intermembrane compartment that makes up approximately 40% of the cell volume .

How should researchers interpret conflicting data between in vitro and in vivo studies of I. hospitalis srp19 function?

When faced with discrepancies between in vitro and in vivo studies of I. hospitalis srp19 function, consider the following interpretation framework:

• Temperature Considerations:

  • In vitro studies may not accurately replicate the 90°C environment where I. hospitalis naturally functions

  • Assess whether experiments were conducted at physiologically relevant temperatures

• Membrane Environment Effects:

  • The unique membrane architecture of I. hospitalis may not be replicated in simplified systems

  • Consider how the absence of the intermembrane compartment might affect protein function in vitro

• Interactome Differences:

  • In vivo, srp19 functions within a complex network of proteins

  • Absence of partner proteins in vitro may alter observed functions

• Methodological Reconciliation:

  • Develop intermediate complexity systems (e.g., reconstituted membranes, cell extracts)

  • Use complementary techniques to bridge the in vitro-in vivo gap

  • Consider native mass spectrometry to capture intact complexes

• Data Integration Approach:

  • Weight evidence based on experimental proximity to native conditions

  • Develop computational models that can reconcile divergent datasets

  • Use Bayesian statistical approaches to integrate conflicting data points

This framework acknowledges the complex cellular architecture of I. hospitalis and the challenges of studying hyperthermophilic proteins outside their native environment.

What bioinformatic approaches are most appropriate for analyzing the evolutionary history of I. hospitalis srp19?

For comprehensive evolutionary analysis of I. hospitalis srp19, implement these specialized bioinformatic approaches:

• Phylogenetic Analysis:

  • Construct maximum likelihood trees using diverse archaeal, bacterial, and eukaryotic srp19 sequences

  • Implement archaeal-specific substitution models that account for amino acid frequency biases

  • Use Bayesian approaches to assess confidence in evolutionary relationships

• Domain Architecture Analysis:

  • Compare domain organization across TACK superphylum archaea

  • Identify lineage-specific insertions or deletions

  • Map functional domains to evolutionary transitions

• Coevolution Analysis:

  • Identify coevolving residues within srp19

  • Analyze coevolution between srp19 and SRP RNA

  • Detect correlated evolutionary changes with other SRP components

• Ancestral Sequence Reconstruction:

  • Infer ancestral srp19 sequences at key evolutionary nodes

  • Synthesize and characterize ancestral proteins

  • Compare functional properties to modern variants

• Horizontal Gene Transfer Assessment:

  • Search for signatures of horizontal gene transfer

  • This is particularly relevant given that I. hospitalis has been shown to have genes of potential bacterial origin

  • Analyze synteny and genomic context across related species

These approaches are particularly valuable since I. hospitalis belongs to the TACK superphylum, which has been proposed as evolutionarily related to the ancestry of eukaryotes .

How can researchers differentiate between direct and indirect effects when studying the impact of N. equitans on I. hospitalis srp19 expression?

Differentiating between direct and indirect effects of N. equitans on I. hospitalis srp19 expression requires a multi-faceted experimental design:

• Time-Course Analysis:

  • Monitor changes in srp19 expression at different stages of N. equitans attachment

  • Early changes are more likely to represent direct effects

  • Establish temporal relationships between different cellular responses

• Spatial Correlation Studies:

  • Use fluorescence in situ hybridization to localize srp19 mRNA

  • Correlate expression changes with proximity to N. equitans attachment sites

  • Map protein localization relative to contact points

• Pathway Inhibition Experiments:

  • Selectively inhibit stress response pathways

  • Block specific signaling cascades to identify mediators

  • Use conditional mutants to interrupt potential intermediate pathways

• Contact-Free Conditioned Media Experiments:

  • Test whether secreted factors from N. equitans can induce changes in srp19 expression

  • Compare with direct contact conditions

  • Filter and fractionate conditioned media to identify specific factors

• Comparative Proteomics:

  • Use approaches similar to those in the research by Giannone et al.

  • Create correlation networks to identify proteins with expression patterns similar to srp19

  • Cluster responses to identify coordinated pathways

This approach builds on previous observations that N. equitans induces significant changes in I. hospitalis protein expression patterns, particularly in membrane biogenesis and post-translational protein modification pathways .

What are the best practices for maintaining recombinant I. hospitalis srp19 stability during purification and storage?

For optimal stability of recombinant I. hospitalis srp19 during purification and storage, implement these evidence-based practices:

• Buffer Optimization:

  • Include 5-10% glycerol to prevent aggregation

  • Maintain pH between 7.0-7.5 to mimic cytoplasmic conditions

  • Test various salt concentrations (200-500 mM) to enhance stability

  • Add reducing agents (1-5 mM DTT or TCEP) to prevent oxidation of cysteine residues

• Temperature Management:

  • Perform purification steps at room temperature rather than 4°C

  • Consider heat treatment (60-70°C) as a purification step to remove E. coli proteins

  • Store protein at -80°C with flash-freezing in liquid nitrogen to prevent freeze-thaw damage

• Stabilizing Additives:

  • Test thermostabilizing compounds like trimethylamine N-oxide (TMAO)

  • Include divalent cations (Mg2+) at physiological concentrations

  • Consider adding small amounts of SRP RNA to stabilize native conformation

• Storage Conditions:

  • Store concentrated protein (>1 mg/ml) in small aliquots

  • Avoid repeated freeze-thaw cycles

  • Test lyophilization with appropriate cryoprotectants as an alternative storage method

• Quality Control Protocol:

  • Verify protein state by dynamic light scattering before and after storage

  • Monitor activity through RNA binding assays

  • Implement regular SDS-PAGE analysis to check for degradation

These practices are particularly important for I. hospitalis proteins, which have evolved to function optimally at 90°C in a hyperthermophilic marine environment .

How can researchers overcome challenges in reconstituting functional I. hospitalis SRP complexes in vitro?

Reconstituting functional I. hospitalis SRP complexes in vitro presents unique challenges due to the hyperthermophilic nature and complex cellular organization of this organism. Address these challenges with the following specialized approach:

• Component Preparation:

  • Express and purify all SRP proteins individually with compatible tags

  • Synthesize SRP RNA using in vitro transcription with thermostable RNA polymerases

  • Verify folding of individual components before assembly attempts

• Assembly Conditions:

  • Test assembly at elevated temperatures (60-90°C) to mimic native conditions

  • Optimize salt concentrations to balance RNA-protein interactions

  • Use step-wise assembly protocols with carefully controlled order of addition

  • Monitor assembly by native gel electrophoresis at each step

• Functional Verification Methods:

  • Develop high-temperature signal sequence binding assays

  • Establish GTPase activity measurements for SRP GTPases

  • Implement ribosome binding assays using thermostable ribosomes

• Membrane Interaction Studies:

  • Create liposomes with lipid compositions mimicking I. hospitalis membranes

  • Consider the unique dual-membrane system of I. hospitalis when designing reconstitution experiments

  • Test both inner and outer membrane compositions separately

• Stabilization Strategies:

  • Add molecular crowding agents to mimic cellular conditions

  • Include specific ions found in the hyperthermophilic marine environment

  • Test the addition of chaperones to facilitate proper complex formation

These approaches acknowledge the complex membrane architecture of I. hospitalis and the potential impact of its hyperthermophilic lifestyle on protein-protein and protein-RNA interactions.

What are the most promising research directions for understanding the role of srp19 in the evolution of the archaeal endomembrane system?

The unique cellular architecture of I. hospitalis, particularly its complex endomembrane system with secretory functions , positions srp19 research at the frontier of understanding archaeal cell biology and evolution. The most promising research directions include:

• Comparative Structural Biology:

  • Determine high-resolution structures of srp19 from diverse archaea

  • Compare with eukaryotic homologs to identify conserved features

  • Investigate how srp19 structure relates to membrane complexity across archaea

• Synthetic Biology Approaches:

  • Create minimal reconstituted systems to test srp19 function

  • Engineer chimeric srp19 proteins combining features from different domains of life

  • Develop in vitro evolution systems to explore alternative srp19 functionalities

• Advanced Imaging Studies:

  • Implement super-resolution microscopy to track srp19 localization

  • Use electron tomography to visualize srp19 complexes in the context of the I. hospitalis endomembrane system

  • Develop in situ labeling approaches compatible with the unique cellular architecture

• Systems Biology Integration:

  • Map the complete interactome of srp19 in I. hospitalis

  • Develop computational models of protein targeting in the dual-membrane system

  • Integrate transcriptomic, proteomic, and structural data into predictive models

• Evolutionary Reconstruction:

  • Resurrect ancestral srp19 proteins to test functional capabilities

  • Investigate how srp19 function correlates with membrane complexity across archaea

  • Test hypotheses about the role of protein targeting in the evolution of cellular compartmentalization

These directions leverage the proposal that the eukaryotic endomembrane system might have originated from archaea, particularly from members of the TACK superphylum like I. hospitalis .

How might CRISPR-Cas9 gene editing technologies be adapted for studying srp19 function in I. hospitalis?

Adapting CRISPR-Cas9 gene editing for I. hospitalis presents unique challenges due to the hyperthermophilic nature and unusual cellular architecture of this organism. A strategic approach should include:

• Thermostable CRISPR Systems:

  • Identify and engineer Cas9 or Cas12 variants from thermophilic organisms

  • Test activity at the 90°C growth temperature of I. hospitalis

  • Optimize guide RNA stability for high-temperature conditions

• Delivery Methods:

  • Develop transformation protocols specific for the dual-membrane architecture

  • Test electroporation parameters optimized for I. hospitalis

  • Explore liposome-based delivery systems compatible with the outer cellular membrane

• Target Design Strategy:

  • Create conditional knockdown rather than knockout of essential genes like srp19

  • Design allelic replacement strategies to introduce point mutations

  • Implement inducible promoter systems for controlled expression

• Phenotypic Analysis Plan:

  • Monitor effects on protein localization to different membrane compartments

  • Assess impact on the endomembrane system using electron microscopy techniques

  • Evaluate consequences for the relationship with N. equitans

• Co-culture Considerations:

  • Develop methods to distinguish direct effects on I. hospitalis from indirect effects on N. equitans

  • Create reporter systems visible in the co-culture context

  • Establish quantitative metrics for assessing the impact on the symbiotic relationship