Recombinant Nitrosomonas europaea UPF0042 nucleotide-binding protein NE1849 (NE1849)

What is the UPF0042 nucleotide-binding protein NE1849?

UPF0042 nucleotide-binding protein NE1849 is a full-length protein from Nitrosomonas europaea (strain ATCC 19718 / CIP 103999 / KCTC 2705 / NBRC 14298). The protein belongs to the UPF0042 family of nucleotide-binding proteins, with a Uniprot accession number of Q82TN5 . The protein consists of 288 amino acids and is believed to play a role in nucleotide binding, though its specific function in Nitrosomonas europaea metabolism is still being characterized .

How should NE1849 be stored to maintain stability and activity?

The stability of recombinant NE1849 is affected by multiple factors including storage temperature, buffer composition, and the protein formulation. For optimal storage:

• Liquid formulations can be stored for approximately 6 months at -20°C/-80°C

• Lyophilized (freeze-dried) preparations maintain stability for up to 12 months at -20°C/-80°C

• For working aliquots, store at 4°C for up to one week

• Repeated freeze-thaw cycles should be avoided as they can cause protein denaturation

• For reconstitution, use deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Addition of 5-50% glycerol (final concentration) is recommended for long-term storage

• The default recommended final concentration of glycerol is 50%

These storage conditions are essential for maintaining the structural integrity and functional properties of the protein during experimental work.

What purification methods yield the highest purity of recombinant NE1849?

While specific purification protocols for NE1849 are not detailed in the literature, effective purification strategies for recombinant nucleotide-binding proteins generally involve:

• Initial capture using affinity chromatography, typically with a tag system such as histidine-tag or GST-tag (the specific tag type for NE1849 is determined during the manufacturing process)

• Secondary purification via ion exchange chromatography to separate based on charge differences

• Size exclusion chromatography as a polishing step to achieve high purity

• Quality assessment via SDS-PAGE, with NE1849 showing >85% purity in typical preparations

The choice of purification strategy should be optimized based on the expression system used and the specific requirements of downstream applications.

How can researchers study the nucleotide-binding properties of NE1849?

To characterize the nucleotide-binding properties of NE1849, researchers can employ several complementary approaches:

• Fluorescence spectroscopy: Using fluorescently labeled nucleotides or intrinsic tryptophan fluorescence to monitor binding events

• Isothermal titration calorimetry (ITC): To determine binding affinity, stoichiometry, and thermodynamic parameters

• Surface plasmon resonance (SPR): For real-time binding kinetics and affinity determination

• Differential scanning fluorimetry: To assess thermal stability shifts upon nucleotide binding

• Crystallography or cryo-EM: To determine the structural basis of nucleotide binding and identify key residues involved in the interaction

The protein sequence contains a GSGK motif (residues 10-13), which is characteristic of a P-loop or Walker A motif found in many nucleotide-binding proteins, suggesting potential GTPase or ATPase activity . This feature should guide the design of functional assays.

What is the potential functional role of NE1849 in Nitrosomonas europaea metabolism?

While the specific function of NE1849 in Nitrosomonas europaea has not been fully characterized, several approaches can help elucidate its role:

• Comparative genomic analysis: Examining the genomic context of NE1849 and comparing with related genes in other organisms

• Transcriptomic studies: Analyzing when and under what conditions NE1849 is expressed

• Gene knockout or knockdown studies: Observing phenotypic effects when NE1849 is absent or reduced

• Protein-protein interaction studies: Identifying binding partners that may indicate pathways in which NE1849 participates

Nitrosomonas europaea is known for its role in ammonia oxidation and nitrogen cycling . Several studies have characterized the transcriptomic response of N. europaea under different growth conditions . Given that NE1849 is a nucleotide-binding protein, it may potentially be involved in signaling pathways, energy metabolism, or regulation of nitrogen metabolism, though experimental validation is required to confirm these hypotheses.

How might NE1849 relate to the nitrogen metabolism of Nitrosomonas europaea?

Nitrosomonas europaea is a chemolithoautotrophic ammonia-oxidizing bacterium that plays a crucial role in the global nitrogen cycle. To investigate potential connections between NE1849 and nitrogen metabolism:

• Expression correlation analysis: Compare expression patterns of NE1849 with known nitrogen metabolism genes under various growth conditions

• Protein localization studies: Determine if NE1849 co-localizes with ammonia oxidation machinery

• Metabolic flux analysis: Measure changes in nitrogen compound processing when NE1849 is overexpressed or knocked down

Research has shown that N. europaea contains multiple gene clusters involved in nitrogen metabolism, including norCBQD that encodes nitric oxide reductase . Studies on N. europaea under oxygen-limited conditions have revealed significant changes in gene expression related to nitrogen metabolism . Investigating whether NE1849 expression changes under similar conditions could provide insights into its potential role in nitrogen cycling.

How can researchers design experiments to study potential interactions between NE1849 and other proteins in Nitrosomonas europaea?

For investigating protein-protein interactions involving NE1849:

• Co-immunoprecipitation (Co-IP): Using antibodies against NE1849 to pull down interacting proteins

• Bacterial two-hybrid systems: For screening potential interactions in vivo

• Pull-down assays: Using recombinant tagged NE1849 as bait

• Proximity-based labeling approaches: Such as BioID or APEX to identify proteins in close proximity to NE1849 in the cellular context

• Crosslinking mass spectrometry: To capture transient interactions and determine interaction interfaces

When designing these experiments, researchers should consider that Nitrosomonas europaea has a complex metabolic network involved in ammonia oxidation and nitrogen cycling . Potential interaction partners might include proteins involved in nitrogen metabolism, stress response, or energy production pathways.

What bioinformatic approaches are most useful for predicting NE1849 function?

Several bioinformatic strategies can be employed to gain insights into NE1849 function:

• Sequence motif analysis: The protein contains a GSGK motif typical of P-loop NTPases, suggesting ATP/GTP binding capability

• Structural homology modeling: Using related proteins with known structures as templates

• Phylogenetic analysis: Examining evolutionary relationships with functionally characterized proteins

• Gene neighborhood analysis: Studying genomic context for functional associations

• Co-expression network analysis: Identifying genes with similar expression patterns

Integrating these approaches can generate hypotheses about NE1849 function that can then be experimentally validated. The UPF0042 family designation (Uncharacterized Protein Family) indicates that this protein belongs to a group whose function has not yet been experimentally determined, making bioinformatic predictions especially valuable for guiding experimental design.

How should researchers analyze contradictory results in NE1849 activity assays?

When faced with inconsistent results in NE1849 functional studies:

• Methodological analysis: Compare experimental conditions, protein preparations, and assay components

• Protein quality assessment: Verify protein folding, stability, and post-translational modifications

• Context-dependent function evaluation: Consider whether NE1849 activity may depend on specific cofactors or conditions present in some experiments but not others

• Multiple technique validation: Confirm findings using orthogonal experimental approaches

• Statistical rigor: Apply appropriate statistical tests and consider sample sizes when evaluating significance of differences

Research on Nitrosomonas europaea metabolism has shown that enzyme activities can vary significantly with growth conditions, particularly oxygen availability . Such environmental factors should be carefully controlled and reported when performing NE1849 activity assays.

What are the key considerations when integrating NE1849 studies into broader research on Nitrosomonas europaea biology?

• Metabolic pathway integration: Consider how NE1849 may function within known metabolic networks

• Environmental relevance: Relate laboratory findings to ecological roles of Nitrosomonas europaea

• Comparative analysis: Examine NE1849 homologs in related nitrifying bacteria

• Systems biology approaches: Integrate transcriptomic, proteomic, and metabolomic data

Nitrosomonas europaea serves important ecological roles in nitrification and nitrogen cycling in natural and engineered systems . Recent research has focused on its responses to environmental stressors and growth conditions . Understanding NE1849 within this broader context will enhance the significance and application of research findings.