Recombinant Hyperthermus butylicus Signal recognition particle 19 kDa protein (srp19)

What is Hyperthermus butylicus and why is its SRP19 protein of interest to researchers?

Hyperthermus butylicus is a hyperthermophilic sulfur-reducing archaebacterium isolated from the seafloor of a solfataric habitat with temperatures of up to 112°C on the coast of the island of São Miguel, Azores. This extraordinary organism grows optimally between 95 and 106°C at a pH of 7.0 and 17 g of NaCl per liter . It belongs to the archaeal kingdom Crenarchaeota, representing a distinct lineage within this major branch of archaebacteria .

The genome of H. butylicus consists of a single circular chromosome of 1,667,163 bp with a 53.7% G+C content. Among its 1,672 annotated genes, 1,602 are protein-coding, and up to a third are specific to H. butylicus . Notably, the genome is remarkably stable, with no detectable transposable or integrated elements, no inteins, and introns exclusive to tRNA genes .

The Signal Recognition Particle 19 kDa protein (SRP19) from H. butylicus is of particular interest because it functions at extremely high temperatures while maintaining critical cellular functions. As a component of the SRP complex, which is responsible for cotranslational targeting of proteins to membranes, studying SRP19 from hyperthermophiles provides unique insights into:

• Molecular adaptations that enable proteins to function at near-boiling temperatures

• Evolutionary conservation of essential cellular machinery across domains of life

• Structural features that contribute to thermostability while preserving function

• Potential biotechnological applications of thermostable proteins in industrial processes

What is the function of SRP19 in cellular processes?

SRP19 plays a crucial role in the assembly and function of the Signal Recognition Particle (SRP), a ribonucleoprotein complex central to protein targeting across all domains of life. Recent research has elucidated several key functions of SRP19:

• RNA binding and SRP assembly: SRP19 binds directly to SRP RNA, inducing conformational changes that facilitate the binding of other SRP proteins, particularly SRP54. This binding is essential for proper assembly of the functional SRP complex .

• Protein targeting: As part of the SRP complex, SRP19 contributes to recognizing signal sequences in nascent polypeptides as they emerge from the ribosome. This recognition is critical for targeting secretory and membrane proteins to the appropriate cellular compartments .

• Cellular viability: Research has demonstrated that SRP19 is a core cell-essential gene required for proliferation in all cell types. CRISPR-mediated knockout of SRP19 across 1,019 cell lines results in cell death in all cases, regardless of SRP19 expression levels . This universal essentiality highlights the fundamental importance of SRP19 in cellular function.

• Development and differentiation: In humans, genetic defects in SRP19 cause severe congenital neutropenia, indicating its critical role in neutrophil granulocyte differentiation . Studies show that SRP19 mutations lead to aberrant splicing and decreased protein expression, disrupting normal neutrophil development .

• Nuclear-cytoplasmic shuttling: In eukaryotes, SRP19 has been observed to localize in nucleoli, nuclei, and cytoplasm. This distribution pattern suggests a dynamic role in SRP assembly that spans multiple cellular compartments .

Understanding the function of SRP19 in hyperthermophilic archaea like H. butylicus provides insights into how this essential cellular machinery has evolved to operate under extreme environmental conditions.

How is recombinant H. butylicus SRP19 typically expressed and purified?

The expression and purification of recombinant H. butylicus SRP19 presents unique challenges due to its origin from a hyperthermophilic organism. Based on methodologies used for similar archaeal proteins, the following approach is recommended:

Expression System Selection and Vector Design:

E. coli remains the preferred host for recombinant expression of archaeal proteins due to its ease of genetic manipulation, rapid growth, and high yields. BL21(DE3) or Rosetta strains are particularly suitable as they accommodate rare codons often present in archaeal genes. For vector design, a codon-optimized H. butylicus SRP19 gene should be cloned into an expression vector with an inducible promoter (typically T7) and an appropriate fusion tag to facilitate purification .

Expression Conditions:

• Induction at OD600 of 0.6-0.8 with 0.5-1.0 mM IPTG

• Post-induction growth at 16-25°C for 16-20 hours (despite H. butylicus being a hyperthermophile, lower expression temperatures often improve proper folding in E. coli)

• Harvesting cells by centrifugation and resuspension in an appropriate buffer (typically phosphate or Tris-based, pH 6.0-8.0)

Purification Strategy:

The thermostability of H. butylicus proteins provides a useful first purification step:

• Heat treatment (70-80°C for 15-30 minutes) of the cell lysate to denature most E. coli host proteins

• Removal of denatured proteins by centrifugation

• Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged proteins

• Size exclusion chromatography to separate monomeric SRP19 from aggregates

• If necessary, ion exchange chromatography as an additional purification step

Quality Assessment:

The purity and identity of the recombinant protein should be confirmed by:

• SDS-PAGE and Western blotting

• Mass spectrometry to verify molecular weight

• Circular dichroism to assess proper folding

• Functional assays to confirm RNA-binding activity

For NMR studies, samples typically require 1.0-2.0 mM purified protein in 50 mM potassium phosphate buffer (pH 6.0) with 90% H2O/10% D2O .

What structural characteristics define H. butylicus SRP19?

While the specific three-dimensional structure of H. butylicus SRP19 has not been fully characterized in the available literature, its structural features can be inferred from related proteins and general principles of hyperthermophilic protein architecture.

RNA-Binding Interface:

The functional interaction between SRP19 and SRP RNA likely involves:

• A positively charged surface created by conserved basic residues (lysine, arginine)

• Aromatic residues that may participate in base-stacking interactions

• A specific binding pocket adapted to recognize structural features of the SRP RNA

Thermostability Features:

As a protein from an organism growing optimally at 95-106°C, H. butylicus SRP19 likely displays several thermostability-enhancing characteristics:

• Increased number of ion pairs and salt bridges

• Higher proportion of charged residues on the protein surface

• Reduced content of thermolabile amino acids (asparagine, glutamine)

• Compact hydrophobic core with optimized packing

• Shorter loop regions and potentially higher secondary structure content

• Specific adaptations that stabilize the protein-RNA interface at high temperatures

Post-Translational Modifications:

While specific modifications of H. butylicus SRP19 have not been extensively characterized, archaeal proteins, including chromatin proteins in Crenarchaeota, can undergo methylation . Such modifications might contribute to the stability or functional properties of H. butylicus SRP19.

Definitive structural characterization would require techniques such as X-ray crystallography, NMR spectroscopy (as described for related proteins in search result ), or cryo-electron microscopy. These approaches would reveal the unique structural adaptations that enable H. butylicus SRP19 to function under extreme conditions.

How does temperature affect the functionality of H. butylicus SRP19?

The functionality of H. butylicus SRP19 is intrinsically linked to temperature, reflecting its origin from an organism that thrives at 95-106°C. Understanding the temperature-function relationship provides insights into both the protein's biological role and potential biotechnological applications.

Optimal Temperature Range:

Given that H. butylicus grows optimally between 95 and 106°C and can survive up to 108°C , its SRP19 protein likely exhibits maximum functionality within this temperature range. This extreme thermostability distinguishes it from mesophilic homologs, which typically denature well below 80°C.

Temperature-Dependent Structural Stability:

H. butylicus SRP19 maintains its native fold at temperatures that would rapidly denature proteins from mesophilic organisms. This extraordinary stability likely derives from:

• Enhanced hydrophobic core packing

• Extensive networks of electrostatic interactions

• Increased structural rigidity in regions not directly involved in function

• Possible temperature-dependent conformational changes that optimize activity

RNA-Binding Characteristics:

The interaction between SRP19 and its target RNA is expected to display unique temperature dependence:

• Stable complex formation at temperatures approaching the boiling point of water

• Potentially altered binding kinetics compared to mesophilic homologs

• Specialized adaptations that maintain specificity under conditions that typically destabilize nucleic acid structures

Physiological Significance:

The temperature profile of H. butylicus SRP19 activity would be expected to align with the organism's growth requirements. This correlation ensures that essential cellular processes, including protein targeting via the SRP pathway, function optimally under the extreme conditions of its natural habitat.

To experimentally characterize these temperature effects, researchers can employ techniques such as:

• Thermal denaturation studies using differential scanning calorimetry or circular dichroism

• Temperature-dependent RNA binding assays

• Functional reconstitution of the SRP complex at varying temperatures

• Structural analyses at different temperatures using NMR or other suitable methods

Understanding the temperature-function relationship of H. butylicus SRP19 provides valuable insights into molecular adaptation strategies and may inspire the design of thermostable proteins for biotechnological applications.

What experimental conditions are optimal for studying the thermostability of recombinant H. butylicus SRP19?

Investigating the extraordinary thermostability of H. butylicus SRP19 requires specialized experimental approaches that accommodate extreme temperature conditions while maintaining analytical precision. The following methodological framework provides optimal conditions for comprehensive characterization:

Buffer Optimization:

The choice of buffer is critical when working at elevated temperatures:

• Temperature-stable buffers: Phosphate buffers maintain pH stability at high temperatures better than Tris-based systems, which have significant temperature coefficients.

• pH adjustment: Remember that pH values change with temperature; buffers should be prepared considering the ΔpKa/°C of the buffering agent.

• Salt concentration: Include physiologically relevant concentrations of NaCl (approximately 17 g/L as found in H. butylicus growth conditions) .

• Stabilizing additives: Consider including 5-10% glycerol or other osmolytes that may enhance thermostability without affecting function.

Thermal Stability Analysis:

A multi-technique approach provides comprehensive insights into thermal stability:

• Differential scanning calorimetry (DSC): Permits direct measurement of melting temperature (Tm) and thermodynamic parameters (ΔH, ΔS, ΔG) up to 130°C.

• Circular dichroism (CD) spectroscopy: Tracks secondary structure changes during thermal denaturation.

• Intrinsic fluorescence: Monitors tertiary structure changes via shifts in tryptophan or tyrosine fluorescence.

• Dynamic light scattering (DLS): Assesses potential aggregation at elevated temperatures.

Activity Assays at Elevated Temperatures:

Functional characterization requires:

• Temperature-controlled RNA binding assays (EMSA, fluorescence anisotropy)

• Specialized equipment capable of maintaining stable temperatures up to 110°C

• Reference measurements using mesophilic SRP19 proteins as controls

• Time-course experiments to distinguish between immediate effects and progressive changes

Experimental Design Considerations:

Working with hyperthermophilic proteins presents unique challenges:

• Evaporation prevention: Use sealed containers or pressure cells for extended incubations.

• Temperature calibration: Ensure precise temperature control and monitoring.

• Specialized equipment: High-temperature cuvette holders, pressure-tolerant cells.

• Statistical validation: Multiple replicates (minimum of three) for each condition.

This table summarizes recommended experimental parameters for thermal stability studies:

By systematically applying these approaches, researchers can characterize the thermostability of H. butylicus SRP19 and identify the molecular features that contribute to its function under extreme conditions.

How can researchers address potential expression challenges of recombinant H. butylicus SRP19 in E. coli or other host systems?

Expression of recombinant proteins from hyperthermophilic archaea in mesophilic hosts presents significant challenges. For H. butylicus SRP19, a systematic approach can overcome these obstacles:

Codon Optimization and Gene Synthesis:

H. butylicus employs a high level of GUG and UUG start codons, contrasting with the standard AUG preference in E. coli . Additionally, the 53.7% G+C content of H. butylicus differs from E. coli:

• Perform comprehensive codon optimization based on E. coli codon usage bias

• Remove rare codons that might cause translational pausing

• Optimize the 5' region to minimize mRNA secondary structure that could impede translation initiation

• Consider synthetic gene construction rather than direct cloning from genomic DNA

Expression Vector Selection:

The choice of expression system significantly impacts success:

• Test multiple fusion partners for enhanced solubility:

  • Small tags: His6, FLAG, Strep-II

  • Solubility-enhancing tags: SUMO, MBP, GST, Trx

• Include a cleavable linker between the tag and SRP19 (e.g., TEV protease site)

• Evaluate different promoter strengths (T7, tac, ara) to optimize expression levels

Host Strain Selection:

Different E. coli strains offer advantages for archaeal protein expression:

• BL21(DE3): Standard expression strain with reduced protease activity

• Rosetta or CodonPlus: Provides rare tRNAs potentially required for archaeal genes

• Arctic Express: Contains cold-adapted chaperonins that may aid folding

• C41/C43: Specifically developed for potentially toxic protein expression

Optimization of Expression Conditions:

A systematic grid of conditions should be tested:

Co-expression Strategies:

If standard approaches yield poor results, consider:

• Co-expression with molecular chaperones (GroEL/ES, DnaK/DnaJ/GrpE)

• Co-expression with other components of the SRP complex that might stabilize SRP19

• Test archaeal-specific chaperones if available

Alternative Expression Systems:

If E. coli proves unsuitable:

• Cell-free expression systems: Allow direct addition of stabilizers and avoid toxicity

• Thermophilic expression hosts (Thermus thermophilus): Better match to natural conditions

• Yeast expression systems: More complex folding machinery that may accommodate archaeal proteins

Refolding Approaches:

For protein expressed in inclusion bodies:

• Solubilize in 6-8 M urea or guanidinium hydrochloride

• Perform stepwise dialysis or rapid dilution for refolding

• Include stabilizing additives (L-arginine, glycerol, sucrose) in refolding buffer

• Test heat treatment during refolding to promote native conformation

By systematically addressing these aspects, researchers can significantly improve the chances of successfully expressing functional recombinant H. butylicus SRP19 protein.

What analytical techniques are most effective for characterizing the RNA-binding properties of H. butylicus SRP19?

Characterizing the RNA-binding properties of H. butylicus SRP19 requires specialized approaches that account for both the protein's extreme thermostability and its specific interactions with SRP RNA. The following analytical techniques provide complementary insights:

Biophysical Binding Assays:

• Electrophoretic Mobility Shift Assay (EMSA):

  • Particularly useful for initial characterization

  • Can detect complex formation at different protein:RNA ratios

  • Should be performed at various temperatures (25-95°C) to assess temperature dependence

  • Requires heat-stable gel systems for high-temperature analyses

• Fluorescence Anisotropy/Polarization:

  • Real-time monitoring of binding using fluorescently labeled RNA

  • Provides accurate Kd measurements in solution

  • Can be performed at different temperatures to derive thermodynamic parameters

  • Requires thermostable fluorophores for high-temperature studies

• Surface Plasmon Resonance (SPR) or Bio-Layer Interferometry (BLI):

  • Measures association and dissociation kinetics (kon and koff)

  • Reveals binding kinetics across temperature ranges

  • Allows testing of binding under various buffer conditions

  • Temperature-controlled flow cells enable high-temperature measurements

• Isothermal Titration Calorimetry (ITC):

  • Direct measurement of binding energetics (ΔH, ΔS, ΔG)

  • No labeling required, avoiding potential interference

  • Provides stoichiometry information

  • High-temperature ITC instruments can operate at temperatures relevant to H. butylicus

Structural Characterization of the Complex:

• Nuclear Magnetic Resonance (NMR) Spectroscopy:

  • Can map the RNA-binding interface by chemical shift perturbation experiments

  • Provides dynamic information at atomic resolution

  • Works well for smaller RNA fragments

  • Can be performed at various temperatures

  • Requires 15N/13C-labeled protein preparation as described in search result

• X-ray Crystallography:

  • Provides high-resolution structures of the protein-RNA complex

  • Reveals precise molecular interactions

  • May require crystallization screening under specialized conditions

• Hydroxyl Radical Footprinting:

  • Identifies RNA regions protected by protein binding

  • Can be performed under varied temperature conditions

  • Provides structural information in solution

Specificity and Functional Analysis:

• RNA Competition Assays:

  • Compare binding to specific vs. non-specific RNA

  • Identify minimal binding motifs

  • Test structural requirements (e.g., stem-loops, bulges)

• Mutational Analysis:

  • Systematically mutate key residues in SRP19 or nucleotides in SRP RNA

  • Correlate sequence changes with binding alterations

  • Identify critical interaction points

This integrated approach enables comprehensive characterization of the unique properties of H. butylicus SRP19-RNA interactions under conditions that approximate its natural hyperthermophilic environment.

How might mutations in conserved residues affect the functionality of H. butylicus SRP19?

Investigating the effects of mutations in conserved residues of H. butylicus SRP19 provides valuable insights into structure-function relationships and evolutionary constraints. A systematic approach to mutation analysis yields the most informative results:

Identification of Conserved Residues:

• Comparative Sequence Analysis:

  • Align H. butylicus SRP19 with homologs from diverse archaea, bacteria, and eukaryotes

  • Identify universally conserved residues across domains of life

  • Identify residues conserved specifically in thermophilic organisms

  • SRP19 is highly conserved functionally, with mutations in humans causing severe congenital neutropenia

• Structural Conservation Mapping:

  • Map conserved residues onto structural models

  • Categorize by location (core, surface, interface)

  • Predict functional roles based on structural context

Rational Design of Mutations:

Different classes of mutations provide complementary insights:

• RNA-Binding Interface Mutations:

  • Basic residues (Arg, Lys) → Ala: Expected to disrupt RNA binding

  • Aromatic residues → Ala: May disrupt base stacking interactions

  • These should be prioritized as SRP19's primary function involves RNA binding

• Thermostability-Related Mutations:

  • Target ion pairs or salt bridges that may contribute to extreme thermostability

  • Introduce residues common in mesophilic homologs

  • Modify surface charge distribution

• Protein-Protein Interface Mutations:

  • Target residues likely involved in interactions with SRP54

  • Mutations in these regions may affect SRP assembly without directly affecting RNA binding

Experimental Characterization:

A comprehensive assessment includes:

• Stability Analysis:

  • Thermal denaturation studies (CD, DSC)

  • Compare wild-type and mutant melting temperatures

  • Assess effects on conformational stability

• RNA Binding Characterization:

  • Quantitative binding assays (EMSA, fluorescence anisotropy)

  • Determine changes in binding affinity (Kd)

  • Assess kinetic parameters (kon, koff)

• Functional Analysis:

  • Reconstitution of partial or complete SRP complex

  • Assessment of SRP assembly efficiency

  • Correlation of molecular effects with functional outcomes

The table below illustrates how different mutations might affect H. butylicus SRP19 functionality:

This systematic mutational analysis can provide insights into how H. butylicus SRP19 achieves the delicate balance between extreme thermostability and maintained functionality, potentially revealing principles applicable to protein engineering.

What insights can H. butylicus SRP19 provide about protein adaptation to extreme environments?

H. butylicus SRP19 represents an excellent model system for understanding protein adaptation to extreme environments. As a component of the essential protein targeting machinery in an organism that thrives at 95-106°C , this protein offers unique perspectives on molecular evolution under extreme conditions:

Comparative Genomic and Evolutionary Insights:

The H. butylicus genome shows remarkable stability with no detectable transposable or integrated elements, no inteins, and introns exclusive to tRNA genes . This genomic stability suggests a relatively constant evolutionary environment, allowing us to study protein adaptations that evolved specifically for extreme thermophily rather than as responses to changing conditions.

• Thermophilic Specialization:

  • H. butylicus contains many genes specific to its lifestyle, with up to a third of its 1,602 protein-coding genes being unique

  • SRP19 adaptation likely represents specialized evolution for an extreme niche

• Evolutionary Conservation vs. Adaptation:

  • SRP19 must maintain core functionality while adapting to extreme conditions

  • The balance between conserved functional regions and adapted structural elements reveals evolutionary constraints

Molecular Mechanisms of Thermostability:

Several strategies likely contribute to H. butylicus SRP19 thermostability:

• Amino Acid Composition Biases:

  • Expected enrichment in charged residues (Arg, Lys, Glu, Asp)

  • Likely reduction in thermolabile residues (Asn, Gln, Cys, Met)

  • Potentially higher frequency of aromatic residues for enhanced hydrophobic packing

• Structural Reinforcement:

  • Enhanced hydrophobic core packing

  • Extensive networks of salt bridges and ion pairs

  • Minimized loop regions susceptible to thermal fluctuations

• RNA-Protein Interface Adaptation:

  • Specialized interactions that remain stable at high temperatures

  • Potentially stronger electrostatic interactions to stabilize nucleic acid binding

Functional Adaptation Principles:

• Temperature-Activity Relationships:

  • Activity likely peaks at temperatures matching physiological conditions (95-106°C)

  • May retain some functionality at lower temperatures, allowing laboratory study

• Stability-Function Trade-offs:

  • Reveals how proteins balance the competing demands of thermostability and maintained function

  • Identifies which regions can be rigidified and which must retain flexibility

• Adaptation to Cellular Environment:

  • SRP19 functions within the context of H. butylicus's cellular environment

  • May depend on specific ionic conditions (e.g., salt concentration of 17 g NaCl/L)

  • Could require interaction with other thermoadapted cellular components

Application to Protein Engineering:

The principles derived from studying H. butylicus SRP19 have broader implications:

• Rational Design of Thermostable Proteins:

  • Identifies transferable features for engineering proteins with enhanced thermostability

  • Reveals which stabilizing strategies maintain functionality

• Biotechnological Applications:

  • Template for designing proteins for high-temperature industrial processes

  • Insights into creating thermostable enzymes for biocatalysis

• Synthetic Biology Approaches:

  • Building blocks for designing biological systems that function under extreme conditions

  • Models for minimal cellular systems with enhanced robustness

The analysis of H. butylicus SRP19 contributes to our fundamental understanding of how essential cellular machinery can be adapted to function under conditions approaching the upper temperature limits of life, while maintaining the precise interactions required for protein targeting and cellular viability.