Recombinant Archaeoglobus fulgidus Uncharacterized protein AF_1301 (AF_1301)

What are the predicted physiochemical properties of AF_1301 and how do they compare to other characterized A. fulgidus proteins?

AF_1301 is an uncharacterized protein from Archaeoglobus fulgidus, an extremophilic archaeon that thrives in high-temperature environments between 60-95°C . When approaching uncharacterized proteins from A. fulgidus, researchers should first examine predicted properties including molecular weight, theoretical isoelectric point (pI), amino acid composition, and predicted domains. Based on studies of other A. fulgidus proteins like AF2331, many proteins from this organism display unusual amino acid compositions that reflect adaptation to extreme conditions .

For instance, the characterized protein AF2331 has a theoretical pI of 4.3 due to an abundance of acidic residues (9 Asp and 13 Glu residues) compared to basic residues . When analyzing AF_1301, researchers should consider whether it exhibits similar acidic or basic surface clusters that might indicate potential protein-protein interactions within operons, as observed with AF2331 and AF2330, which appear to form charge-stabilized complexes . Initial characterization should include sequence analysis tools such as PSI-BLAST to identify potential homologs and threading calculations to predict structural elements that might hint at function.

What are the optimal expression systems for producing recombinant AF_1301 protein?

Recombinant expression of proteins from hyperthermophilic archaea presents unique challenges due to their extreme native environmental conditions. For expressing AF_1301, researchers should consider both prokaryotic (E. coli) and eukaryotic expression systems, with modifications to accommodate the protein's thermophilic origins. E. coli BL21(DE3) containing chaperone plasmids often proves useful for archaeal protein expression, as they can assist with proper folding of these challenging proteins.

When expressing AF_1301, cultivation temperature represents a critical parameter that requires optimization. While A. fulgidus naturally grows at temperatures between 60-95°C with an optimum around 83°C , recombinant expression typically occurs at lower temperatures (15-30°C) to balance protein production with proper folding. Researchers should implement a factorial design exploring various induction temperatures, IPTG concentrations, and induction durations. Additionally, specialized tags (such as SUMO or MBP) often improve solubility of archaeal proteins, though they should be subsequently removed for structural studies to prevent interference with native protein conformation.

How can I optimize buffer conditions for AF_1301 protein stability during purification?

Buffer optimization for AF_1301 should consider the native environmental conditions of Archaeoglobus fulgidus. Given that A. fulgidus is a marine hyperthermophile, buffers containing moderate salt concentrations (300-500 mM NaCl) often enhance protein stability . Since many A. fulgidus proteins exhibit unusual surface charge distributions (as seen with AF2331's negatively charged surface clusters), researchers should test a range of buffer pH values (typically pH 6.0-8.0) to identify optimal conditions for AF_1301 stability .

Temperature stability represents another critical consideration for purification. While working at elevated temperatures that mimic the native environment (60-83°C) might improve stability for some applications, most purification steps are performed at lower temperatures (4-25°C) for practical reasons . Researchers should empirically determine if AF_1301 requires additives such as glycerol (10-20%), reducing agents (DTT or β-mercaptoethanol), or specific divalent cations (Mg²⁺, Ca²⁺) that might stabilize the protein structure. Given that A. fulgidus grows under high hydrostatic pressure conditions in its native environment, stability tests at various pressures might provide insights into optimal handling conditions for maintaining activity during purification .

What crystallization strategies are most effective for obtaining diffraction-quality crystals of AF_1301?

Crystallization of proteins from hyperthermophilic archaea like Archaeoglobus fulgidus often requires specialized approaches due to their unusual physicochemical properties. Based on successful crystallization of other A. fulgidus proteins such as AF2331 (which was solved by Se-Met MAD to 2.4 Å resolution), researchers should implement sparse matrix screening with commercial kits specifically designed for thermophilic proteins . These initial screens should explore various precipitants (PEG variants, ammonium sulfate), pH ranges (4.0-9.0), and salt concentrations.

Temperature represents a critical variable for crystallization of proteins from thermophiles. While standard crystallization typically occurs at 4-20°C, researchers should also attempt crystallization at elevated temperatures (30-60°C) that better reflect the protein's native environment without reaching the optimal growth temperature of A. fulgidus (83°C) . Seeding techniques often prove particularly valuable for archaeal proteins, as they can overcome nucleation barriers that prevent crystal formation. If molecular replacement is not feasible due to lack of suitable structural homologs (as was the case with AF2331), researchers should prepare selenomethionine-labeled protein for experimental phasing methods such as MAD or SAD .

How can I determine if AF_1301 forms oligomeric structures similar to other A. fulgidus proteins?

Many proteins from Archaeoglobus fulgidus, including the characterized AF2331, form interesting oligomeric assemblies with unusual intersubunit interactions . To investigate potential oligomerization of AF_1301, researchers should employ a multi-method approach beginning with size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine absolute molecular weight in solution. Native PAGE analysis provides complementary evidence, though researchers should note that unusual charge distributions (common in A. fulgidus proteins) might affect migration patterns .

Analytical ultracentrifugation (AUC) offers more definitive characterization of oligomeric states, particularly sedimentation velocity experiments to identify distinct species in solution. For high-resolution analysis of subunit interactions, researchers should consider small-angle X-ray scattering (SAXS) to generate low-resolution molecular envelopes of AF_1301 in solution. If structural data becomes available, researchers should examine potential oligomerization interfaces for unusual features such as the interdigitated interactions observed in AF2331, which represents a novel type of fold with domain swapping between subunits . Cross-linking mass spectrometry can further identify specific residues involved in subunit interfaces if initial studies confirm oligomerization.

What approaches can reveal the fold classification of AF_1301 if it represents a novel structural motif?

Determining whether AF_1301 represents a novel protein fold requires comprehensive structural analysis similar to what was performed for AF2331, which was ultimately classified as a new type of α + β fold . Researchers should first perform thorough computational analyses using multiple structural comparison tools including CE, DALI, SSM, and TM-Align to search for structural similarities against the entire PDB database . The absence of significant matches across multiple algorithms would suggest a potentially novel fold, as was demonstrated for AF2331 where the highest TM-score achieved was only 0.48 (below the 0.5 threshold typically used to define common folds) .

If crystallographic or NMR structures are obtained, detailed topological analysis should examine the arrangement and connectivity of secondary structure elements. Researchers should pay particular attention to unusual structural features such as the interdigitated interactions observed in AF2331 that contribute to its unique fold classification . For objective classification, quantitative metrics such as TM-score are preferable to subjective assessment of structural similarity. Given that many A. fulgidus proteins adapt to extreme conditions through unique structural arrangements, researchers should consider how thermostability might influence the evolution of novel folds specific to this extremophile.

What bioinformatic approaches can predict potential functions of AF_1301?

Functional prediction for uncharacterized proteins like AF_1301 requires integrating multiple computational approaches to compensate for the limitations of individual methods. Genomic context analysis provides critical insights, as demonstrated with AF2331, which was found to be encoded in the same operon as the basic protein AF2330, suggesting a potential functional relationship between these proteins . Researchers should examine neighboring genes and operon structure of AF_1301 using tools like STRING to identify functional associations that might indicate involvement in specific metabolic pathways relevant to A. fulgidus' unique physiology.

How can I design experiments to test the involvement of AF_1301 in sulfur metabolism pathways of A. fulgidus?

Given that Archaeoglobus fulgidus is a sulfur-metabolizing organism with unique sulfate reduction capabilities, uncharacterized proteins like AF_1301 might play roles in these distinctive metabolic pathways . To investigate this possibility, researchers should design knockout or knockdown experiments targeting the AF_1301 gene, followed by phenotypic characterization focusing on growth rates under various sulfur metabolism conditions (sulfate reduction, thiosulfate reduction) at different pressures (0.3-60 MPa) and temperatures (optimally 83°C) .

In vitro biochemical assays should test direct interaction with sulfur compounds by incubating purified recombinant AF_1301 with potential substrates and monitoring changes through spectrophotometric methods or chromatography. Protein-protein interaction studies using pull-down assays or bacterial two-hybrid systems can identify associations with known components of sulfur metabolism pathways in A. fulgidus. Researchers should perform these experiments under conditions that reflect the organism's native environment, including elevated temperatures (83°C) and potentially high pressure conditions, as A. fulgidus exhibits different growth patterns under heterotrophic versus autotrophic growth conditions at varying pressures .

What approaches can determine if AF_1301 is involved in high-pressure adaptation of A. fulgidus?

Recent studies have shown that Archaeoglobus fulgidus exhibits piezophilic or piezotolerant behavior depending on its metabolic strategy, growing at pressures up to 60 MPa heterotrophically and 40 MPa autotrophically . To investigate whether AF_1301 contributes to pressure adaptation, researchers should compare expression levels of the AF_1301 gene under various pressure conditions using qRT-PCR or RNA-seq analysis of A. fulgidus cultured at different pressures (0.3-60 MPa) . Significant upregulation at elevated pressures would suggest potential involvement in pressure adaptation mechanisms.

Protein stability and activity assays with purified recombinant AF_1301 should be conducted under various pressure conditions using specialized high-pressure equipment similar to that used for A. fulgidus cultivation studies . Researchers should examine whether the protein exhibits enhanced stability or altered activity profiles at elevated pressures that would indicate evolutionary adaptation to high-pressure environments. Structural studies comparing protein conformations at ambient versus high pressure could reveal pressure-sensitive regions that might function as molecular switches. If genetic manipulation systems are available for A. fulgidus, creating AF_1301 knockout strains and examining their growth characteristics under various pressure conditions would provide definitive evidence of its role in pressure adaptation .

How does AF_1301 compare to homologous proteins in other extremophiles?

Comparative analysis of AF_1301 with proteins from other extremophiles can provide evolutionary insights and functional clues. Researchers should construct comprehensive phylogenetic trees including homologs (if identified) from other archaeal extremophiles, particularly those adapted to similar environmental stresses. Special attention should be paid to organisms with different combinations of adaptations (thermophilic but not piezophilic, or vice versa) to isolate specific adaptive features associated with each environmental factor.

Sequence conservation analysis can identify residues under positive or negative selection pressure that might indicate functional importance. Researchers should compare predicted or determined structural features of AF_1301 with characterized proteins from other extremophiles, looking for common adaptation strategies such as increased hydrophobic core packing, additional salt bridges, or disulfide bonds that contribute to thermostability. Given the unique interdigitated structural motifs observed in other A. fulgidus proteins like AF2331, researchers should examine whether similar unusual structural arrangements exist in homologous proteins from other extremophiles as potential convergent adaptations to extreme environments .

What can computational simulations reveal about AF_1301's stability under extreme conditions?

Molecular dynamics (MD) simulations represent a powerful approach for investigating the behavior of proteins like AF_1301 under the extreme conditions experienced by Archaeoglobus fulgidus. Researchers should conduct comparative MD simulations of AF_1301 models or structures at various temperatures (ambient vs. 83°C) and pressures (ambient vs. 20-60 MPa) to identify structural elements that contribute to stability under these extreme conditions . These simulations should analyze metrics including root-mean-square deviation (RMSD), radius of gyration, hydrogen bond networks, and salt bridge dynamics over the trajectory.

How can I overcome solubility issues when working with recombinant AF_1301?

Proteins from hyperthermophilic archaea like Archaeoglobus fulgidus frequently present solubility challenges when expressed in mesophilic hosts due to their adaptation to extreme environmental conditions. When encountering solubility issues with recombinant AF_1301, researchers should first attempt expression at lower temperatures (15-18°C) with reduced inducer concentrations to slow protein production and facilitate proper folding. Solubility-enhancing fusion partners such as MBP, SUMO, or thioredoxin often dramatically improve solubility of archaeal proteins.

Researchers should optimize lysis buffer conditions based on the characteristics of other A. fulgidus proteins. For instance, if AF_1301 contains multiple charged residues similar to AF2331 (which has numerous acidic residues on its surface), buffers with appropriate ionic strength and pH should be selected to maintain solubility . Addition of stabilizing agents such as arginine (50-100 mM), proline, or specific osmolytes (trehalose, glycerol) can significantly enhance solubility. If these approaches prove insufficient, researchers should consider on-column refolding protocols or extraction from inclusion bodies using mild detergents or chaotropic agents followed by step-wise dialysis to remove these agents while maintaining protein solubility.

What special considerations apply when analyzing the activity of AF_1301 at high temperatures?

Activity assays for proteins from hyperthermophiles like Archaeoglobus fulgidus require modifications to standard protocols to accommodate their temperature optima. When designing activity assays for AF_1301, researchers should develop thermostable buffer systems that maintain pH at elevated temperatures, as standard buffers often exhibit significant pH shifts at the high temperatures (83°C) where A. fulgidus proteins function optimally . Equipment calibration is essential, as temperature gradients within reaction vessels can lead to inconsistent results.

Researchers should implement appropriate controls to distinguish thermal denaturation of assay components from actual enzymatic activity. Reference proteins with known thermostability profiles should be included as controls in parallel experiments. If spectrophotometric methods are employed, researchers must account for temperature effects on absorption coefficients and increased evaporation rates. When comparing activities across temperature ranges, Arrhenius plots should be constructed to determine activation energies rather than simply reporting activities at individual temperatures. If AF_1301 shows potential pressure adaptations similar to A. fulgidus' growth characteristics, specialized equipment allowing combined high-temperature and high-pressure measurements might be necessary for physiologically relevant activity assessment .

How might AF_1301 contribute to biotechnological applications based on A. fulgidus' extremophilic properties?

Proteins from extremophiles like Archaeoglobus fulgidus represent valuable biocatalysts for industrial processes requiring stability under harsh conditions. If functional characterization reveals enzymatic activity in AF_1301, researchers should evaluate its potential applications in high-temperature industrial processes where conventional enzymes rapidly denature. The protein's thermostability profile should be thoroughly characterized, determining both its temperature optimum and half-life at various elevated temperatures ranging from 60-95°C to match the growth range of A. fulgidus .

The unusual structural features observed in other A. fulgidus proteins, such as the novel α + β fold of AF2331, highlight the potential for discovering unique structural arrangements in AF_1301 that might inspire protein engineering approaches . If AF_1301 demonstrates pressure adaptation (similar to A. fulgidus' ability to grow at pressures up to 60 MPa), researchers should investigate applications in high-pressure biocatalysis or as a model for studying pressure effects on protein structure and function . Beyond direct applications, fundamental insights from studying AF_1301 could contribute to our understanding of protein adaptation mechanisms to extreme environments, informing the design of stabilized proteins for various biotechnological applications.

How can cryo-EM approaches complement crystallographic studies of AF_1301?

Cryo-electron microscopy (cryo-EM) offers complementary structural information to crystallography, particularly valuable for challenging proteins from extremophiles like Archaeoglobus fulgidus. For AF_1301, researchers should consider cryo-EM when crystallization proves difficult or to validate crystallographic structures obtained under non-native conditions. Single-particle cryo-EM is especially useful if AF_1301 forms oligomeric assemblies similar to the interdigitated dimeric structure observed for AF2331, as it can reveal conformational heterogeneity that might be averaged out in crystal structures .

Sample preparation for AF_1301 cryo-EM studies should explore various buffer conditions to prevent protein aggregation on grids. Grid preparation at elevated temperatures might better represent the protein's native conformation, though technical limitations of current cryo-EM workflows must be considered. For proteins with novel folds like those found in A. fulgidus, integrative structural approaches combining crystallography, cryo-EM, and computational modeling often provide the most complete structural insights . If AF_1301 interacts with other proteins (like the hypothesized interaction between AF2331 and AF2330), cryo-EM can visualize these complexes without requiring co-crystallization, potentially revealing physiologically relevant interactions that contribute to A. fulgidus' adaptation to extreme environments .