Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_2124 (AF_2124)

What is known about the basic properties of Archaeoglobus fulgidus protein AF_2124?

AF_2124 is an uncharacterized protein from the hyperthermophilic archaeon Archaeoglobus fulgidus DSM 4304. Based on computational structure prediction models, the protein has a relatively high confidence score (pLDDT global: 88.13) , suggesting a well-defined tertiary structure. The protein has been identified in the genome of A. fulgidus, but its specific function remains to be fully elucidated. As part of an extremophile organism capable of growing at temperatures around 83°C and pressures up to 40-60 MPa , AF_2124 may possess thermostable and barostable properties that make it interesting for structural and functional studies.

What are the optimal growth conditions for Archaeoglobus fulgidus for maximal protein expression?

Archaeoglobus fulgidus exhibits optimal growth at 83°C under various pressure conditions. For heterotrophic metabolism (lactate oxidation coupled to sulfate reduction), maximum growth rates are observed at 20 MPa, showing moderate piezophilic behavior. For autotrophic metabolism (CO₂ fixation coupled to thiosulfate reduction via H₂), growth rates remain nearly constant from 0.3 to 40 MPa, indicating piezotolerance . When cultivating A. fulgidus for protein extraction, these conditions should be considered:

These conditions can be used for native protein expression before extraction for further characterization .

Which expression system is most suitable for producing recombinant AF_2124?

E. coli remains the preferred expression system for recombinant archaeal proteins due to its ease of genetic manipulation, rapid growth, and high protein yields. The T7 promoter system in pET vectors (medium copy number with pMB1 origin) is particularly effective for thermostable proteins from archaea, potentially yielding target protein representing up to 50% of total cell protein in successful cases .

For AF_2124 expression, consider these optimized parameters:

For thermostable proteins like AF_2124, post-induction expression at lower temperatures can significantly improve protein solubility while maintaining the intrinsic stability of the native protein .

How can codon optimization enhance the expression of AF_2124 in heterologous systems?

Codon optimization is critical when expressing archaeal proteins in bacterial systems due to differences in codon usage biases. For AF_2124, which comes from A. fulgidus with a high GC content and different codon preferences compared to E. coli, optimization should follow these methodological guidelines:

• Analyze the codon adaptation index (CAI) of the native sequence

• Replace rare codons with synonymous codons frequently used in the host organism

• Avoid creating mRNA secondary structures or internal Shine-Dalgarno sequences

• Maintain a balanced GC content throughout the sequence

Comparative expression studies have shown that codon optimization for archaeal genes can increase protein yield by 5-15 fold in E. coli systems. Additionally, optimizing the 5' region of the transcript is particularly important for translation initiation efficiency .

What computational methods can predict the structure and function of AF_2124?

The structure of AF_2124 has been computationally modeled with high confidence (pLDDT global: 88.13) , indicating reliable prediction. Researchers can employ the following computational approaches for deeper structural and functional predictions:

• Sequence-Based Methods:

  • Profile-sequence and profile-profile comparisons using HHpred

  • Conserved domain analysis through CDD, SMART, and Pfam

  • Evolutionary coupling analysis to identify co-evolving residues

• Structure-Based Methods:

  • Structural alignment with characterized proteins using DALI or TM-align

  • Binding site prediction with CASTp, POCASA, or FTSite

  • Molecular dynamics simulations to evaluate stability at high temperatures and pressures

• Integrative Approaches:

  • Combined sequence conservation and surface topology analysis

  • Genomic context analysis to identify functional associations

  • AlphaFold2-based predictions refined with molecular dynamics simulations

These methods can provide initial hypotheses about AF_2124 function that can guide experimental design for biochemical characterization .

What are the challenges in crystallizing hyperthermophilic archaeal proteins like AF_2124?

Crystallizing proteins from hyperthermophilic archaea presents unique challenges but also advantages. For AF_2124, consider these methodological approaches:

• Challenges and Solutions:

  • High salt requirements: Screen various salt types and concentrations (0.2-3.0 M)

  • Hydrophobic surface patches: Use amphiphilic additives like detergents (0.1-0.5% w/v)

  • Structural flexibility: Employ surface entropy reduction mutations

  • Oxidation-sensitive cysteines: Maintain reducing conditions with 1-5 mM DTT or TCEP

• Thermostability Advantages:

  • Use higher crystallization temperatures (20-30°C) for more ordered crystal formation

  • Exploit intrinsic stability for longer crystallization trials

  • Test ligand or substrate co-crystallization to stabilize functional conformations

• Recommended Screening Approach:

  • Initial broad screening at three temperatures (4°C, 20°C, 30°C)

  • Secondary optimization focusing on pH ranges 5.5-8.5

  • Micro-batch under oil and vapor diffusion methods in parallel

  • Addition of physiologically relevant ions (particularly zinc if it contains cysteine clusters like other A. fulgidus proteins)

How can transcriptomic data inform experimental approaches to characterize AF_2124?

Transcriptomic studies of A. fulgidus under stress conditions can provide valuable insights into the potential function of AF_2124. Based on whole-genome microarray studies of heat shock response in A. fulgidus, approximately 14% of open reading frames show altered transcript abundance under stress conditions .

To leverage transcriptomic data for AF_2124 characterization:

• Expression Pattern Analysis:

  • Compare AF_2124 expression profiles under various stressors (heat, pressure, oxidative stress)

  • Identify co-expressed genes for potential functional relationships

  • Determine if AF_2124 is part of a stress response operon

• Regulatory Element Identification:

  • Look for conserved motifs in the promoter region similar to known regulatory elements

  • Check for palindromic motifs like CTAAC-N5-GTTAG found in heat shock response genes

  • Analyze potential interaction with transcriptional regulators like HSR1

• Experimental Design Based on Transcriptomic Data:

  • Focus functional assays on conditions where AF_2124 is highly expressed

  • Consider knockout/complementation experiments based on co-expression networks

  • Investigate protein-protein interactions with co-expressed gene products

This approach connects genomic, transcriptomic, and functional data to build a comprehensive understanding of the protein's biological role .

What experimental approaches can determine if AF_2124 contains zinc-binding motifs similar to other A. fulgidus proteins?

Some A. fulgidus proteins, such as DNA polymerase D (Pol-D), contain critical zinc-binding cysteine clusters that are essential for function . To investigate if AF_2124 has similar structural features:

• Sequence Analysis:

  • Identify conserved cysteine clusters in the AF_2124 sequence

  • Compare pattern to known zinc-binding motifs (C-X₂-C-X₂₄-C-X₂-C)

  • Perform multiple sequence alignment with characterized zinc-binding proteins

• Experimental Metal Content Determination:

  • Inductively coupled plasma mass spectrometry (ICP-MS) of purified protein

  • Colorimetric zinc detection using 4-(2-pyridylazo)resorcinol (PAR)

  • Atomic absorption spectroscopy for quantitative metal analysis

• Functional Validation:

  • Site-directed mutagenesis of predicted zinc-coordinating residues

  • Activity assays with and without zinc supplementation

  • Metal chelation studies using EDTA or 1,10-phenanthroline

  • Circular dichroism spectroscopy to assess structural changes upon metal binding/removal

If AF_2124 contains zinc-binding motifs, this could indicate roles in DNA binding, catalysis, or structural stability at high temperatures .

How does AF_2124 compare to similar uncharacterized proteins in other extremophilic archaea?

Comparative genomic analysis can reveal evolutionary relationships and potential functional conservation of AF_2124 across different archaeal species:

• Phylogenetic Analysis:

  • Construct phylogenetic trees using maximum likelihood methods

  • Calculate evolutionary distances between homologs

  • Identify orthologous relationships across archaeal lineages

• Conservation Patterns:

  • Analyze selective pressure using dN/dS ratios

  • Identify ultra-conserved residues as potential functional sites

  • Map conservation onto the predicted 3D structure

• Genomic Context Conservation:

  • Compare gene neighborhoods across species

  • Identify conserved operonic structures

  • Analyze synteny between A. fulgidus and related species

The comparison with the genomic context of AF_2124 across the five Archaeoglobus species genomes available (including strain 7324 from North Sea oil fields) could reveal functional associations based on conserved gene neighborhoods .

What insights can comparative genomics provide about the role of AF_2124 in A. fulgidus adaptation to extreme environments?

Comparative genomics approaches can illuminate how AF_2124 might contribute to A. fulgidus' remarkable adaptability to high temperature and pressure environments:

• Distribution Analysis:

  • Map the presence/absence of AF_2124 homologs across extremophiles with varying optima

  • Correlate protein sequence features with organism habitat parameters

  • Compare strains isolated from different extreme environments (like strain 7324 from oil fields versus VC-16 from marine hydrothermal vents)

• Structural Adaptation Signatures:

  • Analyze amino acid composition biases associated with thermostability

  • Identify stabilizing interactions unique to extremophile homologs

  • Compare charged residue distribution on protein surfaces

• Molecular Evolution Rates:

  • Calculate evolutionary rates in thermophiles versus mesophiles

  • Identify accelerated evolution in specific domains

  • Analyze positive selection signatures in extremophile lineages

The core genome analysis of Archaeoglobus species identified 1001 core genes, with AF_2124 potentially being part of the core or accessory genome that contributes to the specific adaptations of these hyperthermophiles .

How can high-hydrostatic pressure cultivation techniques be applied to study AF_2124 function in native-like conditions?

To study AF_2124 under conditions that mimic its native deep-sea environment, researchers can employ high-hydrostatic pressure (HHP) cultivation techniques:

• Methodological Approach:

  • Use static high-pressure vessels with specialized sampling systems

  • Employ glass syringes with minimal headspace for optimal pressure control

  • Implement proper decompression protocols (~19 MPa/min) to minimize stress responses

• Experimental Design:

  • Compare AF_2124 expression levels across a pressure gradient (0.3 to 60 MPa)

  • Analyze protein modifications under different pressure conditions

  • Examine localization and interaction partners at optimal versus stress pressures

• Analytical Techniques:

  • Quantitative proteomics to measure AF_2124 abundance across conditions

  • Protein extraction under pressure-maintaining conditions

  • In situ activity assays under maintained pressure

This approach would reflect the true physiological conditions experienced by A. fulgidus in deep marine environments (2-5 km below sea level, 20-50 MPa pressures) and could reveal pressure-dependent functions of AF_2124.

What experimental design strategies can resolve contradictory data when characterizing novel proteins like AF_2124?

When characterizing novel proteins like AF_2124, researchers often encounter contradictory data that requires careful experimental design to resolve:

• Sources of Contradictory Data:

  • Expression system artifacts versus native function

  • In vitro versus in vivo activity discrepancies

  • Computational predictions conflicting with experimental results

• Robust Experimental Design Approach:

  • Employ factorial design to systematically test multiple variables (temperature, pressure, pH, salt concentration)

  • Use randomized block design to control for confounding variables

  • Implement controls for system-specific artifacts

• Statistical Analysis for Resolution:

  • Apply functional data analysis (FDA) for continuous measurement data

  • Conduct meta-analysis of multiple experimental approaches

  • Use Bayesian methods to integrate prior knowledge with new data

• Confirmation Strategies:

  • Test function in multiple heterologous systems

  • Complement deletion mutants with wild-type and mutant variants

  • Perform in vivo crosslinking to validate interaction partners

A completely randomized experimental design can help control for environmental variables when testing AF_2124 function across different conditions, ensuring that observed effects are genuinely related to the protein's activity .

How can genomic context and co-expression networks inform the design of functional studies for AF_2124?

Genomic context and co-expression networks provide powerful frameworks for designing targeted functional studies:

• Genomic Context Analysis:

  • Identify operonic structures containing AF_2124

  • Analyze nearby regulatory elements and their response conditions

  • Examine conservation of gene neighborhoods across related species

• Co-expression Data Integration:

  • Analyze transcriptomic data across various stress conditions

  • Build weighted gene co-expression networks

  • Identify transcriptional modules containing AF_2124

• Functional Inference and Validation:

  • Design assays based on functions of co-expressed genes

  • Test interactions with predicted partner proteins

  • Investigate shared regulatory mechanisms with co-expressed genes

• Experimental Design Based on Network Predictions:

  • Target conditions where the co-expression module is activated

  • Perform affinity purification coupled with mass spectrometry (AP-MS)

  • Validate protein-protein interactions using bimolecular fluorescence complementation

This systems biology approach can narrow down potential functions from thousands of possibilities to a manageable set of testable hypotheses, significantly accelerating the functional characterization process.

What computational approaches can identify potential DNA-binding activity in AF_2124?

If sequence analysis suggests AF_2124 may have DNA-binding capabilities (similar to HSR1 from A. fulgidus ), these computational and experimental approaches can confirm this function:

• Computational DNA-Binding Prediction:

  • Search for helix-turn-helix, zinc finger, or other DNA-binding motifs

  • Apply machine learning algorithms trained on known DNA-binding proteins

  • Perform electrostatic surface potential mapping to identify positive patches

  • Use molecular docking to predict DNA-protein interactions

• Structural Comparison:

  • Compare with HSR1 (AF1298) which has a helix-turn-helix DNA binding motif

  • Analyze structural similarity to known DNA-binding domains from thermophiles

  • Identify conserved residues in potential DNA-recognition regions

• Binding Site Prediction:

  • Locate clusters of conserved basic residues on the protein surface

  • Predict binding energy landscapes for DNA-protein interactions

  • Model conformational changes upon DNA binding

This integrated computational approach can generate testable hypotheses about specific DNA sequences that might interact with AF_2124, similar to the CTAAC-N5-GTTAG motif recognized by HSR1 .

How can functional screening libraries be designed to identify substrates or binding partners of AF_2124?

Designing comprehensive screening approaches for identifying the function of uncharacterized proteins requires multiple complementary strategies:

• Activity-Based Screening:

  • Design a substrate library based on metabolic pathways in A. fulgidus

  • Screen for enzymatic activities (hydrolase, transferase, oxidoreductase)

  • Monitor cofactor consumption (ATP, NAD(P)H, etc.) in potential reactions

  • Use untargeted metabolomics to identify reaction products

• Protein Interaction Screening:

  • Construct a genomic library of A. fulgidus proteins for two-hybrid screening

  • Perform protein microarray analysis with purified AF_2124

  • Use pull-down assays coupled with mass spectrometry

  • Employ proximity labeling in heterologous systems

• Nucleic Acid Binding Screening:

  • Design SELEX (Systematic Evolution of Ligands by Exponential Enrichment) experiments

  • Perform genomic SELEX to identify native DNA/RNA targets

  • Use electrophoretic mobility shift assays with genomic fragments

  • Conduct ChIP-seq in heterologous systems expressing AF_2124

These comprehensive screening approaches can systematically test thousands of potential functions to identify the biological role of AF_2124.

What methods are effective for determining temperature and pressure stability profiles of AF_2124?

Characterizing the stability of proteins from extremophiles requires specialized techniques that can operate under extreme conditions:

• Thermal Stability Assessment:

  • Differential Scanning Calorimetry (DSC) with extended temperature range (up to 120°C)

  • Circular Dichroism (CD) spectroscopy with high-temperature cells

  • Intrinsic fluorescence monitoring during thermal denaturation

  • Activity retention assays after thermal challenge

• Pressure Stability Techniques:

  • High-pressure spectroscopy using specialized pressure cells

  • Pressure perturbation calorimetry

  • Activity measurements under various pressures using custom pressure vessels

  • FTIR spectroscopy under pressure to monitor secondary structure

• Combined Pressure-Temperature Stability:

  • Phase diagrams construction using multiple biophysical techniques

  • Determination of optimal and limiting conditions for structural integrity

  • Identification of pressure-temperature compensation effects

• Data Analysis and Interpretation:

  • Fitting to appropriate thermodynamic models

  • Calculation of activation energies for unfolding

  • Determination of volume changes during pressure denaturation

These specialized approaches reveal how proteins like AF_2124 maintain structural integrity under the extreme conditions where A. fulgidus thrives (temperatures around 83°C and pressures up to 40-60 MPa) .

How can site-directed mutagenesis be used to identify residues critical for AF_2124 thermostability?

Site-directed mutagenesis represents a powerful approach to understanding the molecular basis of thermostability in proteins like AF_2124:

• Target Selection Strategy:

  • Focus on charged residues in surface loops

  • Target proline residues in turn regions

  • Examine ion pair networks and salt bridges

  • Investigate hydrophobic packing in the protein core

  • Analyze unique amino acid compositions (increased Glu, Lys, reduced Gln, Asn)

• Systematic Mutagenesis Approach:

  • Create alanine scanning libraries of selected regions

  • Engineer charge reversal mutations in ion pair networks

  • Introduce destabilizing mutations found in mesophilic homologs

  • Create chimeric proteins with mesophilic counterparts

• Stability Analysis Pipeline:

  • Thermal denaturation monitoring by CD spectroscopy

  • Differential scanning fluorimetry (Thermofluor) for high-throughput screening

  • Activity retention after thermal challenge

  • Structural analysis of successful stabilizing mutations