Recombinant Natronomonas pharaonis Uncharacterized protein NP1057A (NP1057A)

What is known about the basic properties of the NP1057A protein from Natronomonas pharaonis?

NP1057A is an uncharacterized protein from the extremophilic archaeon Natronomonas pharaonis, strain DSM 2160 / ATCC 35678. The protein consists of 73 amino acids with the sequence MVEIQTGLRENTVGAVEQFAEIASIDPLVASMILSGAIIITVAVVAFGALTLGAIGASIKRGLSGSEEPNQPA, as identified in the recombinant protein records . The gene encoding this protein is designated as NP1057A (ordered locus name) or NP1057E (ORF name), with UniProt accession number Q3IUT9 . Based on the amino acid sequence, particularly the hydrophobic stretch "MILSGAIIITVAVVAFGALTLGAIGA," NP1057A likely functions as a membrane protein, which would be consistent with the archaeon's need for specialized membrane adaptations in extreme environments. This protein is part of the 2,843 protein-coding genes identified in the N. pharaonis genome through rigorous evaluation of automatic gene finder data .

How does the genomic context of NP1057A relate to Natronomonas pharaonis adaptation to extreme environments?

NP1057A exists within the genomic context of N. pharaonis, an extremely haloalkaliphilic archaeon isolated from soda lakes with pH values as high as 11 . The organism's genome consists of a 2.6-Mb GC-rich chromosome and two plasmids (131 kb and 23 kb), with the chromosome having a high G+C content of 63.4% . The genome encodes adaptations for surviving dual extreme conditions: high salinity (3.5 M NaCl optimal growth) and alkaline pH (optimal growth at pH 8.5) . These adaptations include numerous proteins with a high proportion of acidic amino acids (average 19.3%), resulting in low isoelectric points (average pI 4.6) – a characteristic feature of haloarchaea employing the salt-in strategy for hypersaline adaptation . While the specific role of NP1057A remains uncharacterized, its presence in the N. pharaonis genome suggests it may contribute to the organism's remarkable adaptability to extreme conditions, potentially participating in membrane functions critical for maintaining cellular homeostasis in high-salt, high-pH environments.

What are the recommended storage and handling conditions for recombinant NP1057A protein in laboratory settings?

Recombinant NP1057A protein requires specific storage and handling protocols to maintain stability and functionality. The protein should be stored in a Tris-based buffer with 50% glycerol, specifically optimized for this protein . For short-term storage, the protein should be kept at -20°C, while for extended storage periods, conservation at either -20°C or -80°C is recommended . It is crucial to avoid repeated freezing and thawing cycles, as this can lead to protein degradation and loss of activity . For ongoing experiments, it is advisable to prepare working aliquots that can be stored at 4°C for up to one week, minimizing freeze-thaw stress on the protein . Given that NP1057A originates from an extremophilic organism adapted to high salt concentrations and alkaline pH, researchers should consider whether maintaining similar conditions (high salt, alkaline pH) during experimental work might be necessary for proper protein folding and activity, especially for functional studies that aim to characterize this protein's biological role.

How might researchers design experiments to elucidate the functional role of NP1057A in Natronomonas pharaonis?

Elucidating the function of NP1057A requires a multifaceted experimental approach combining genetic, biochemical, and physiological methods. Gene knockout or CRISPR-Cas9 based editing of the NP1057A gene in N. pharaonis would provide insights into the protein's essentiality and phenotypic consequences of its absence . Researchers should compare growth curves, morphology, and stress responses between wild-type and NP1057A-deficient strains under varying salt concentrations and pH values to determine if the protein contributes to extremophilic adaptations. Localization studies using fluorescently-tagged NP1057A would confirm its predicted membrane association and potentially reveal specific subcellular localization patterns. Pull-down assays and co-immunoprecipitation followed by mass spectrometry could identify protein interaction partners, placing NP1057A within cellular networks. Biochemical assays testing potential enzymatic activities should be considered, although the protein's small size (73 amino acids) suggests it may function as a structural component or regulator rather than an enzyme . Transcriptomic and proteomic analyses comparing expression under different environmental conditions (varying salt, pH, nutrient availability) would indicate when NP1057A is most highly expressed, providing clues to its function. These experiments should account for N. pharaonis' unusual physiology, including its proton-based (rather than sodium-based) bioenergetics, which distinguishes it from other alkaliphiles .

How might comparative genomics approaches contribute to understanding the evolutionary significance of NP1057A in halophilic archaea?

Comparative genomics provides a powerful approach for contextualizing NP1057A within the evolutionary landscape of halophilic archaea. Researchers should conduct comprehensive homology searches using both sequence-based (BLAST, HMM profiles) and structure-based prediction methods across archaeal genomes, with particular focus on the Halobacteriaceae family . Such analyses would reveal whether NP1057A represents a conserved archaeal protein or a specialized adaptation unique to N. pharaonis or closely related species. Synteny analysis examining the genomic context of NP1057A homologs across species could identify conserved gene neighborhoods, suggesting functional associations and potential operonic structures. Phylogenetic reconstruction of NP1057A evolutionary history, calibrated using the established archaeal phylogeny based on 16S rRNA and the 53 marker proteins identified in the GTDB database, would reveal patterns of selection acting on this gene . Researchers should pay particular attention to possible horizontal gene transfer events, which are common in archaea and could explain unique adaptations to extreme environments. Comparing the amino acid composition of NP1057A with homologs from non-halophilic archaea might reveal signatures of adaptation to high salt, such as an enrichment in acidic residues – a characteristic adaptation observed in the N. pharaonis proteome (average 19.3% acidic amino acids) . These comparative approaches should be integrated with experimental data on protein function to develop a comprehensive understanding of how NP1057A contributes to the remarkable environmental adaptations of halophilic archaea.

What role might NP1057A play in the unique bioenergetics of Natronomonas pharaonis compared to other alkaliphiles?

NP1057A may have significant implications for the distinctive bioenergetic mechanisms of N. pharaonis, which notably diverges from other alkaliphiles in its energy conversion strategies. Unlike other alkaliphilic bacteria and archaea that utilize sodium ions (Na+) as the coupling ion between respiratory chain and ATP synthase, N. pharaonis has been experimentally proven to use protons (H+) for this purpose . This is particularly intriguing given the bioenergetic challenges of maintaining a proton gradient in alkaline environments. Given NP1057A's putative membrane localization (suggested by its hydrophobic amino acid stretches) and small size (73 amino acids), it could potentially function as a regulatory component of proton transport systems, an accessory protein for respiratory complexes, or a structural element maintaining membrane integrity under extreme pH conditions . Researchers investigating this hypothesis should employ patch-clamp techniques or reconstituted proteoliposome systems containing purified NP1057A to test for ion transport or channel activities. Protein-protein interaction studies focusing on known components of the N. pharaonis respiratory chain could reveal whether NP1057A physically associates with these complexes. Comparative expression analysis under varying bioenergetic conditions (aerobic vs. anaerobic, different carbon sources) would indicate whether NP1057A expression correlates with specific bioenergetic states. Understanding NP1057A's potential role in N. pharaonis bioenergetics could provide insights into novel mechanisms for energy conservation in extreme environments and potentially inform biotechnological applications in bioenergy production under alkaline conditions.

How does the amino acid composition of NP1057A compare to other membrane proteins in N. pharaonis, and what might this reveal about its adaptation to extreme environments?

The amino acid composition of NP1057A provides significant insights into its potential structural adaptations to extreme haloalkaline environments. Analyzing the 73-amino acid sequence (MVEIQTGLRENTVGAVEQFAEIASIDPLVASMILSGAIIITVAVVAFGALTLGAIGASIKRGLSGSEEPNQPA), researchers should quantify the prevalence of acidic residues (Asp, Glu), basic residues (Arg, Lys, His), and hydrophobic amino acids, comparing these frequencies to the proteome-wide averages for N. pharaonis membrane proteins . While the N. pharaonis proteome generally has a high proportion of acidic amino acids (average 19.3%) resulting in low isoelectric points (average pI 4.6) as an adaptation to high internal salt concentrations, membrane proteins often display distinctive compositional biases reflecting their specialized functions at the interface between the cell and its extreme environment . The hydrophobic segment "MILSGAIIITVAVVAFGALTLGAIGA" in NP1057A suggests a transmembrane domain, and researchers should analyze its hydrophobicity profile using scales specifically calibrated for archaeal membrane proteins. Computational modeling and molecular dynamics simulations of NP1057A in membranes mimicking archaeal composition (including archaeol and caldarchaeol lipids) under high salt and high pH conditions would reveal how the protein interacts with its native membrane environment. Comparative analysis with membrane proteins from non-extremophilic archaea would highlight specific adaptations in NP1057A that might contribute to stability and function in extreme conditions. Additionally, researchers should investigate post-translational modifications that might affect protein-membrane interactions, as N. pharaonis is known to employ various glycosylated cell surface proteins to form a protective cell envelope in its harsh native environment .

What specialized protocols should be employed when conducting protein-protein interaction studies with NP1057A in high-salt, alkaline conditions?

Investigating protein-protein interactions involving NP1057A requires methodologies specifically adapted to maintain protein stability in high-salt, alkaline conditions that mimic N. pharaonis' native environment. Traditional co-immunoprecipitation protocols should be modified to include buffers containing 2-3.5 M NaCl and pH 8.5, matching the organism's optimal growth conditions . Cross-linking approaches using membrane-permeable agents like formaldehyde or DSP (dithiobis(succinimidyl propionate)) can capture transient interactions that might otherwise dissociate during cell lysis or purification steps. For in vitro binding assays, researchers should use recombinant NP1057A with potential binding partners expressed and purified under conditions that preserve their native folding, which may require archaeal expression systems rather than bacterial ones . Surface plasmon resonance (SPR) or microscale thermophoresis studies should employ buffers that maintain the high-salt, alkaline conditions throughout the experiment. Yeast two-hybrid systems are generally unsuitable due to their inability to handle extreme conditions, making bacterial two-hybrid or split-protein complementation assays in modified halophilic hosts more appropriate. For membrane-associated interactions, specialized techniques like FRET (Fluorescence Resonance Energy Transfer) in archaeal lipid nanodisc systems or microscopy-based methods using fluorescently tagged proteins in halophilic hosts would provide insights into interactions occurring within the membrane environment. Mass spectrometry-based interaction studies should incorporate specialized sample preparation methods to account for the high salt, which can interfere with ionization, possibly including desalting steps immediately before analysis while maintaining protein complex integrity.

What bioinformatic approaches would be most effective for predicting the function of NP1057A based on its sequence and genomic context?

A comprehensive bioinformatic strategy for predicting NP1057A function should integrate multiple computational approaches, beginning with advanced sequence analysis. Beyond standard homology searches, researchers should employ profile-based methods like PSI-BLAST, HHpred, and HMMER to detect remote homologs that might not be identified with conventional BLAST searches . Structural prediction using the latest deep learning algorithms (AlphaFold2, RoseTTAFold) can generate high-confidence 3D models, revealing potential binding sites or functional domains not evident from sequence alone. Transmembrane topology predictions using algorithms specifically trained on archaeal membrane proteins would confirm the membrane-spanning regions suggested by the hydrophobic stretches in the sequence . Genomic context analysis should examine gene neighborhood conservation across multiple archaeal genomes, as functionally related genes often cluster together and are co-regulated. Researchers should utilize archaeal-specific gene co-expression networks, if available, to identify genes whose expression patterns correlate with NP1057A across different conditions. Protein-protein interaction prediction algorithms could suggest potential binding partners, particularly among other membrane proteins involved in bioenergetics or stress response. Subcellular localization prediction tools calibrated for archaeal proteins would further support or refine the membrane localization hypothesis. These computational analyses should be integrated with the experimental knowledge of N. pharaonis physiology, particularly its unique proton-coupled bioenergetics and adaptations to high salt and pH, to develop testable hypotheses about NP1057A function that can guide subsequent laboratory investigations .

How can researchers effectively design small molecule screening assays to identify compounds that interact with NP1057A for structure-function studies?

Designing effective small molecule screening assays for NP1057A interactions requires careful consideration of the protein's extreme environment adaptation and likely membrane association. Researchers should first establish a stable, functional form of recombinant NP1057A in a system that maintains its native conformation, potentially incorporating it into nanodiscs or liposomes composed of archaeal lipids to mimic its natural membrane environment . Thermal shift assays (differential scanning fluorimetry) modified for high-salt conditions can serve as an initial screening method, identifying compounds that alter protein stability through binding. For membrane proteins like NP1057A, surface plasmon resonance (SPR) assays with the protein immobilized in a supported lipid bilayer would enable real-time detection of binding events while maintaining the protein in a membrane-like environment. Researchers should develop fluorescence-based assays if NP1057A contains tryptophan residues whose fluorescence properties might change upon ligand binding, or alternatively introduce fluorescent labels at strategic positions that don't interfere with function. Compound libraries should include molecules known to interact with archaeal membrane proteins, ion channels, or transporters, focusing on compounds stable under high-salt, alkaline conditions. Molecular docking studies guided by structural predictions can help prioritize compounds for experimental testing. Following identification of binding partners, researchers should validate interactions using orthogonal methods such as isothermal titration calorimetry (ITC) adapted for high-salt conditions, or NMR spectroscopy for mapping binding sites. These assays should be conducted under conditions mimicking N. pharaonis' native environment (3.5 M NaCl, pH 8.5) to ensure physiological relevance of identified interactions .

How might NP1057A function within the broader context of Natronomonas pharaonis adaptation to nitrogen limitation in alkaline environments?

NP1057A's potential role in nitrogen metabolism adaptation merits investigation, given N. pharaonis' remarkable ability to thrive in nitrogen-limited alkaline environments. Genome analysis of N. pharaonis reveals a complete assimilatory nitrate reduction pathway, with the presence of genes nasAB and nirA that enable the organism to utilize nitrate as a nitrogen source - a crucial adaptation to environments where ammonia availability is severely limited due to high pH . While NP1057A is not directly annotated as part of this pathway, its putative membrane localization could implicate it in nitrogen compound transport or sensing mechanisms. Researchers should investigate whether NP1057A expression is regulated in response to varying nitrogen availability, particularly under different nitrate/nitrite/ammonia concentrations. Comparative transcriptomic and proteomic analyses of N. pharaonis under nitrogen-replete versus nitrogen-limited conditions would reveal whether NP1057A is co-regulated with known nitrogen metabolism genes. Additionally, N. pharaonis possesses a complete dissimilatory nitrate reduction pathway to ammonia, distinguished from several other Natronomonas species which have only partial pathways . This suggests specialized adaptations for nitrogen cycling that may involve membrane components like NP1057A in substrate transport or environmental sensing. Integration of NP1057A into nitrogen metabolism models should consider the organism's high degree of nutritional self-sufficiency, as confirmed by its ability to grow in minimal medium with leucine as the sole amino acid supplement . Protein-protein interaction studies specifically targeting known components of nitrogen assimilation and dissimilation pathways could establish direct functional connections between NP1057A and these essential metabolic processes.

What methodological approaches would be most effective for studying the potential role of NP1057A in Natronomonas pharaonis stress response systems?

Investigating NP1057A's potential involvement in stress response systems requires an integrated methodological approach combining physiological, molecular, and systems biology techniques. Researchers should first establish baseline expression patterns of NP1057A under optimal growth conditions (3.5 M NaCl, pH 8.5) using quantitative PCR and western blotting with antibodies against the recombinant protein . Subsequently, N. pharaonis cultures should be subjected to various stressors relevant to its natural environment, including salt concentration fluctuations (both upshock and downshock), pH variations, oxidative stress, UV radiation, and nutrient limitation, followed by time-course analysis of NP1057A expression at both mRNA and protein levels. Global transcriptomic and proteomic analyses under these stress conditions would reveal whether NP1057A is co-regulated with known stress response genes, potentially placing it within specific stress response pathways. Researchers should generate knockout or knockdown strains of NP1057A (if the gene is not essential) and compare their stress tolerance profiles to wild-type N. pharaonis, particularly examining growth rates, survival percentages, and morphological changes under various stress conditions. Fluorescently tagged NP1057A could be used to track potential changes in subcellular localization during stress responses through live-cell imaging techniques adapted for halophilic archaea. Membrane integrity assays before and after stress exposure, comparing wild-type and NP1057A-deficient strains, would indicate whether the protein contributes to maintaining membrane stability under stress conditions. These approaches should be integrated with computational modeling of stress response networks in N. pharaonis, incorporating existing knowledge about signal transduction and motility genes, which are known to be well-represented in this organism's genome .