Recombinant Aquifex aeolicus Uncharacterized protein aq_1894 (aq_1894)

What is known about the uncharacterized protein aq_1894 from Aquifex aeolicus?

The protein aq_1894 is encoded in the genome of Aquifex aeolicus, a hyperthermophilic bacterium that grows optimally at 95°C. Similar to other proteins from this organism, aq_1894 is likely to possess thermostable properties that make it interesting for both basic research and potential biotechnological applications. As an uncharacterized protein, its precise function, structure, and biochemical properties remain to be determined through experimental approaches. Studies of other A. aeolicus proteins have revealed unique adaptations to extreme temperatures, making aq_1894 a candidate for investigating thermostability mechanisms and potentially novel enzymatic activities .

What expression systems are most suitable for recombinant production of aq_1894?

Based on successful expression of other A. aeolicus proteins, several expression systems can be considered:

• E. coli-based systems: Most commonly, E. coli BL21(DE3) or its derivatives are used for thermophilic protein expression. For aq_1894, vectors like pET101/D-TOPO or pBADC3 with C-terminal affinity tags (His-tag or Strep-Tag II) can be employed .

• Expression conditions: Typically, induction with IPTG (for T7-based systems) at OD600 = 0.7-0.8, followed by growth at 37°C for 3-4 hours has been successful for other A. aeolicus proteins .

• Codon optimization: Given the different codon usage between A. aeolicus and E. coli, codon optimization of the aq_1894 sequence may improve expression yields.

• Solubility enhancement: Fusion partners such as MBP (maltose-binding protein) or SUMO can be considered if initial expression attempts yield insoluble protein.

The choice of expression system should be determined by the intended downstream applications and the specific properties of aq_1894.

What purification strategies are recommended for recombinant aq_1894?

A multi-step purification strategy is recommended:

• Initial capture: Affinity chromatography using the engineered tag (Strep-Tag II or His-tag) is effective as a first step. For Strep-tagged protein, Strep-Tactin resin with desthiobiotin elution (5 mM) in an appropriate buffer has been successful for other A. aeolicus proteins .

• Secondary purification: Size-exclusion chromatography (SEC) using a Superdex 200 column to separate oligomeric states and remove aggregates.

• Buffer considerations: Purification buffers should contain stabilizing agents. For thermophilic proteins, this typically includes:

  • 20-50 mM Tris-HCl or HEPES, pH 7.5-8.0

  • 100-300 mM NaCl

  • 5-10% glycerol

  • Potentially 1-5 mM DTT or 2-mercaptoethanol

  • For membrane-associated proteins, appropriate detergents (0.02-0.05% DDM or DM)

• Quality control: Assessment by SDS-PAGE, N-terminal sequencing, and mass spectrometry to confirm protein identity and purity.

Thermostability of A. aeolicus proteins can be advantageous during purification, as a heat treatment step (70-80°C for 10-15 minutes) can be used to remove less stable E. coli proteins, potentially simplifying the purification process .

What approaches should be used to determine the oligomeric state of aq_1894?

Determining the oligomeric state of aq_1894 requires a multi-technique approach:

• Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS):

  • This technique provides accurate molecular weight determination independent of shape

  • Can differentiate between monomers, dimers, and higher-order oligomers

  • Suitable running buffer: 20 mM HEPES pH 7.5, 150 mM NaCl, with appropriate detergent if membrane-associated

• Analytical ultracentrifugation (AUC):

  • Both sedimentation velocity and equilibrium experiments provide information on mass and shape

  • Can detect multiple oligomeric species in a mixture

• Native PAGE and Blue Native PAGE:

  • Provides a quick assessment of oligomeric states

  • BN-PAGE with Coomassie G-250 is particularly useful for membrane proteins

• Cross-linking studies:

  • Using agents like glutaraldehyde or BS3 followed by SDS-PAGE analysis

  • Mass spectrometry analysis of cross-linked products can identify interaction interfaces

• Laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS):

  • Particularly useful for thermophilic protein complexes

  • Can detect native oligomeric states with minimal perturbation

Recent studies of A. aeolicus proteins have revealed unexpected oligomeric arrangements, such as the trimeric structure of Heme A Synthase, despite predictions of monomeric states. This suggests that aq_1894 should be carefully analyzed, as computational predictions may not accurately reflect its native oligomeric state .

What structural characterization methods are most appropriate for aq_1894?

A comprehensive structural characterization strategy includes:

For aq_1894, a combination of these methods would provide complementary structural information, with the choice depending on protein size, stability, and expression yields.

How can the thermostability of aq_1894 be accurately measured and compared to mesophilic homologs?

Multiple complementary approaches should be used to characterize thermostability:

• Differential scanning calorimetry (DSC):

  • Directly measures the heat capacity change during thermal unfolding

  • Provides accurate Tm (melting temperature) values

  • Can detect multiple transitions in multi-domain proteins

  • Recommended temperature range for A. aeolicus proteins: 25-120°C

• Thermal shift assays (TSA)/Differential scanning fluorimetry (DSF):

  • Uses fluorescent dyes (SYPRO Orange) that bind to hydrophobic regions exposed upon unfolding

  • High-throughput method suitable for buffer/additive screening

  • Equipment: Real-time PCR machines with temperature ranges up to 95-100°C

• Circular dichroism (CD) spectroscopy with temperature ramping:

  • Monitors changes in secondary structure during thermal denaturation

  • Can provide information on structural transitions before complete unfolding

• Activity assays at varying temperatures:

  • If enzymatic activity is identified, measuring activity across a temperature range (30-100°C)

  • Determine temperature optima and activation/inactivation kinetics

• Limited proteolysis at different temperatures:

  • Incubation with proteases (trypsin, chymotrypsin) at various temperatures

  • Analysis by SDS-PAGE to assess structural integrity

When comparing to mesophilic homologs, consider:

Similar A. aeolicus proteins have shown remarkable thermostability, with trimeric complexes remaining stable up to 70°C even in the presence of SDS, indicating that aq_1894 may display similar properties .

What computational approaches can predict potential functions of aq_1894?

A comprehensive computational analysis includes:

• Sequence-based analysis:

  • PSI-BLAST and HHpred for remote homology detection

  • PFAM domain identification

  • Conservation analysis across thermophiles and mesophiles

  • Phylogenetic patterns searches (as used for THEP1 identification)

• Structural prediction:

  • AlphaFold2 or RoseTTAFold for 3D structure prediction

  • Structure-based function prediction tools (COFACTOR, COACH)

  • Binding site prediction (CASTp, SiteMap)

• Molecular dynamics simulations:

  • Analyze stability at high temperatures (90-100°C)

  • Identify rigid and flexible regions

  • Compare simulated dynamics with mesophilic homologs

• Genomic context analysis:

  • Examine neighboring genes in the A. aeolicus genome

  • Identify potential operons or functional associations

  • STRING database for predicted functional partners

• Comparative analysis with characterized proteins:

  • Focus on COG (Clusters of Orthologous Groups) classification

  • Examine distribution across thermophiles vs. mesophiles

  • Use this information to design targeted functional assays

Previous studies on uncharacterized thermophile-specific proteins (such as THEP1/COG1618) successfully identified NTPase activity through such computational predictions followed by biochemical validation .

What factorial experimental design would be most appropriate for optimizing aq_1894 expression?

A systematic factorial design approach is recommended for optimizing expression:

• Key factors to consider in a 2^n factorial design:

  • Temperature (28°C vs. 37°C)

  • Induction time point (early log vs. mid-log phase)

  • Inducer concentration (e.g., 0.1 mM vs. 1 mM IPTG)

  • Media composition (LB vs. TB)

  • Co-expression with chaperones (with vs. without)

• Example of a 2^3 factorial design for the three most critical factors:

• Analysis of factorial design results:

  • ANOVA to identify significant main effects and interactions

  • Generation of response surface models for optimization

  • Selection of conditions for validation and scale-up

• Considerations specific to thermophilic proteins:

  • Lower expression temperatures may improve folding despite the protein's thermophilic nature

  • Codon optimization may be necessary due to the GC-rich nature of A. aeolicus genes

  • Consider heat shock (42°C for 30 min) prior to harvesting to activate heat shock chaperones

This structured approach allows for efficient identification of optimal expression conditions while minimizing the number of experiments needed .

What are the best approaches to screen for potential enzymatic activities of aq_1894?

Given the uncharacterized nature of aq_1894, a comprehensive activity screening strategy is recommended:

• Sequence-based function predictions:

  • Use computational predictions to guide initial assays

  • Focus on activities common to the predicted structural fold

• High-throughput activity screens:

  • NTPase/ATPase activity: As observed in other thermophile-specific proteins like THEP1

    • Malachite green phosphate detection assay

    • Luciferase-based ATP consumption assay

    • Test various NTPs (ATP, GTP, CTP, UTP) as substrates

  • Generic enzyme class screens:

    • Hydrolase activity (esterase, protease, glycosidase)

    • Oxidoreductase activity (with various electron acceptors)

    • Transferase activity (with common cofactors)

• Thermal adaptation considerations:

  • Perform assays at various temperatures (37°C, 60°C, 80°C, 95°C)

  • Include appropriate thermostable controls

  • Use buffers with high thermal stability (HEPES, phosphate)

• Activity assay conditions matrix:

• Substrate screening approaches:

  • Commercial substrate libraries

  • Metabolite extracts from A. aeolicus or related organisms

  • Focused substrate sets based on genomic context

Lessons from THEP1 (COG1618) characterization show that even proteins predicted as one enzyme class (nucleotide kinases) may actually function differently (NTPases), highlighting the importance of broad screening approaches .

How can I design experiments to investigate the effects of temperature on aq_1894 structure and function?

A comprehensive approach to investigate temperature effects includes:

• Structural stability analysis across temperature range:

  • Circular dichroism spectroscopy:

    • Far-UV (190-260 nm) for secondary structure

    • Temperature ramps from 25°C to 100°C

    • Calculate the melting temperature (Tm)

  • Intrinsic fluorescence spectroscopy:

    • Monitor changes in tryptophan/tyrosine environments

    • Excitation at 280 nm, emission scan 300-400 nm

    • Perform at 10°C increments from 25-95°C

  • Dynamic light scattering:

    • Monitor size distribution at increasing temperatures

    • Detect onset of aggregation or dissociation of oligomers

• Functional analysis at different temperatures:

  • Enzymatic activity measurements:

    • Determine temperature optimum (Topt)

    • Calculate activation energy (Ea) using Arrhenius plots

    • Compare catalytic efficiency (kcat/Km) across temperatures

  • Ligand binding assays:

    • Isothermal titration calorimetry at various temperatures

    • Calculate thermodynamic parameters (ΔH, ΔS, ΔG)

• Within-subject experimental design for temperature studies:

  • Use the same protein preparation across all temperatures

  • Include temperature ramping (up and down) to test for irreversible changes

  • Implement appropriate statistical methods for correlated measurements

• Data analysis and interpretation:

• Molecular mechanisms of thermostability:

  • Comparative analysis with mesophilic homologs at various temperatures

  • Identification of specific adaptations (ionic interactions, hydrophobic packing)

  • Targeted mutagenesis to test the contribution of specific residues

This experimental design allows for a comprehensive understanding of how temperature affects both structural and functional properties of aq_1894 .

What are common issues when working with recombinant thermophilic proteins and how can they be addressed?

Researchers commonly encounter several challenges when working with thermophilic proteins:

• Low expression yields:

  • Problem: Codon bias between A. aeolicus and E. coli

  • Solution: Use codon-optimized synthetic genes or specialized strains (Rosetta)

  • Problem: Protein toxicity to host

  • Solution: Use tight expression control (pBAD system) or lower temperatures

• Protein insolubility/inclusion bodies:

  • Problem: Improper folding at mesophilic temperatures

  • Solution: Express at lower temperatures (18-25°C) for longer periods

  • Problem: Hydrophobic surface patches

  • Solution: Add solubility tags (MBP, SUMO) or co-express with chaperones

• Purification challenges:

  • Problem: Co-purification of heat shock proteins

  • Solution: Include ATP/MgCl₂ wash steps to dissociate chaperones

  • Problem: Unexpected oligomerization

  • Solution: Screen various buffer conditions with dynamic light scattering

• Activity measurement difficulties:

  • Problem: Standard assay equipment limited to <60°C

  • Solution: Pre-equilibrate reagents, use thermostable coupled enzymes, metal heating blocks

  • Problem: Buffer incompatibility at high temperatures

  • Solution: Use thermostable buffers (phosphate, HEPES) with pH adjusted for temperature shift

• Unexpected post-translational modifications:

  • Problem: E. coli may not reproduce native modifications

  • Solution: Mass spectrometry characterization, alternative expression systems

Each of these challenges has been encountered with other A. aeolicus proteins and can be systematically addressed through careful experimental design and troubleshooting .

How can contradictory results between computational predictions and experimental data for aq_1894 be reconciled?

When facing contradictions between predictions and experimental results:

• Systematic approach to resolve discrepancies:

  • Document all contradictions precisely

  • Evaluate the reliability of both prediction algorithms and experimental methods

  • Design experiments specifically targeting the contradiction

• Common sources of contradiction and solutions:

• Case study from other A. aeolicus proteins:

  • THEP1 (COG1618) was predicted to be a "nucleotide kinase" but experimentally determined to be an NTPase

  • Heme A Synthase was predicted to be monomeric but found to form trimers experimentally

• Resolution strategy:

  • Apply multiple computational approaches

  • Use a range of experimental techniques

  • Consider protein-specific contexts (thermophilic nature, genomic context)

  • Test hypotheses about why predictions might fail (e.g., unique adaptations to high temperature)

• Data integration approach:

  • Weight evidence based on methodological strength

  • Develop models that accommodate both computational and experimental insights

  • Design targeted experiments to test specific hypotheses

Successful resolution of such contradictions can lead to novel insights about thermophilic protein adaptations and improve computational prediction methods for extremophile proteins .

What are the most promising future research directions for aq_1894?

Based on current knowledge of A. aeolicus proteins, several promising research directions emerge:

• Functional characterization:

  • Systematic screening for enzymatic activities, especially focusing on thermophile-specific metabolic pathways

  • Investigation of potential roles in stress response or adaptation to extreme environments

  • Exploration of interaction partners through pull-down assays and proteomics

• Structural biology:

  • High-resolution structure determination through X-ray crystallography or cryo-EM

  • Conformational dynamics studies at different temperatures

  • Structure-guided mutagenesis to identify functional residues

• Evolutionary significance:

  • Comparative analysis across the thermophile-mesophile spectrum

  • Investigation of potential horizontal gene transfer events

  • Reconstruction of ancestral sequences to understand evolutionary adaptations

• Biotechnological applications:

  • Exploration of thermostability mechanisms for protein engineering

  • Potential applications in high-temperature bioprocesses

  • Structure-based design of thermostable biocatalysts

• Integration with systems biology:

  • Transcriptomic and proteomic studies of A. aeolicus under various stress conditions

  • Metabolomic analysis to identify potential substrates or products

  • Computational modeling of metabolic networks including aq_1894

The characterization of aq_1894 has significance beyond this specific protein, as it could reveal general principles of protein thermostability and extremophile adaptation mechanisms .

How can collaborative approaches enhance research on uncharacterized proteins like aq_1894?

Effective collaborative strategies include:

• Interdisciplinary collaboration framework:

  • Computational biologists: Provide predictions and models to guide experiments

  • Structural biologists: Determine 3D structures and dynamics

  • Biochemists: Characterize enzymatic activities and biochemical properties

  • Evolutionary biologists: Analyze phylogenetic distribution and evolutionary history

  • Systems biologists: Place the protein in broader cellular context

• Technology-sharing approaches:

  • Access to specialized equipment for high-temperature experiments

  • Shared resources for expression and purification optimization

  • Collaborative screening platforms for functional characterization

• Data integration strategies:

  • Centralized databases for thermophilic protein properties

  • Standardized protocols and reporting formats

  • Integrated analysis platforms combining multiple data types

• Structured research design:

  • Factorial experimental designs for multi-parameter optimization

  • Coordinated parallel investigations of related proteins

  • Systematic comparison with characterized proteins from the same organism

• Knowledge dissemination and community engagement:

  • Open access publication of results and methods

  • Sharing of recombinant constructs and protocols

  • Regular workshops focusing on extremophile proteins

Successful characterization of proteins like THEP1 and Heme A Synthase from A. aeolicus demonstrates the value of collaborative approaches combining computational predictions, structural analysis, and biochemical characterization .