Recombinant Dictyostelium discoideum Uncharacterized transmembrane protein DDB_G0284989 (DDB_G0284989)

What is DDB_G0284989 protein and what organism does it come from?

DDB_G0284989 is an uncharacterized transmembrane protein from the social amoeba Dictyostelium discoideum, a eukaryotic organism widely used as a model system in biomedical research. This protein, with UniProt ID Q54NT8, is classified as a transmembrane protein, suggesting it spans the cell membrane . Dictyostelium discoideum has been employed as a research model for nearly a century due to its unique life cycle that includes both unicellular and multicellular phases, making it valuable for studying various cellular and developmental processes .

What is the amino acid sequence and structure of DDB_G0284989?

The full-length DDB_G0284989 protein consists of 71 amino acids with the following sequence:

MYKDYLFKSNKGYLSLTLVTLPVCSSLHCYFLWTTLSRLSSLPIDVPRSVCSVASLDLDLVIINLLSILRD

While the detailed three-dimensional structure has not been fully characterized, sequence analysis indicates it is a transmembrane protein. The protein's small size (71 amino acids) suggests it may have a relatively simple structure with potentially a single transmembrane domain. Researchers working with this protein should consider protein structure prediction tools and possibly experimental approaches such as circular dichroism (CD) spectroscopy or nuclear magnetic resonance (NMR) to elucidate its structural features.

How is DDB_G0284989 classified in protein databases?

In protein databases, DDB_G0284989 is classified as an uncharacterized transmembrane protein from Dictyostelium discoideum with the UniProt ID Q54NT8 . The "uncharacterized" designation indicates that the protein's specific function has not been experimentally determined. The protein is also identified by its DDB_G0284989 gene ID in the Dictyostelium genome database. As research progresses, this classification may be updated to reflect new knowledge about the protein's function or structural characteristics.

What are the optimal storage conditions for recombinant DDB_G0284989 protein?

The recombinant DDB_G0284989 protein should be stored at -20°C or -80°C upon receipt, with aliquoting necessary for multiple uses to avoid repeated freeze-thaw cycles that could compromise protein integrity . The protein is typically provided as a lyophilized powder in a Tris/PBS-based buffer containing 6% trehalose at pH 8.0 . For long-term storage, it is recommended to add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) and store the aliquoted protein at -20°C or -80°C . Working aliquots can be stored at 4°C for up to one week, but repeated freezing and thawing should be avoided to maintain protein functionality .

How should recombinant DDB_G0284989 be reconstituted for experimental use?

For reconstitution of recombinant DDB_G0284989, the vial should first be briefly centrifuged to bring the contents to the bottom. The lyophilized protein should then be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL . For long-term storage of the reconstituted protein, glycerol should be added to a final concentration of 5-50% (with 50% being the default recommendation), and the solution should be aliquoted to avoid repeated freeze-thaw cycles . The specific reconstitution protocol may need to be optimized depending on the downstream applications, such as functional assays, structural studies, or antibody production.

What expression systems can be used to produce recombinant DDB_G0284989?

The choice should be based on the research requirements, considering factors such as yield, post-translational modifications, and downstream applications.

What purification methods are most effective for DDB_G0284989?

The search results indicate that recombinant DDB_G0284989 has been produced with an N-terminal His tag , suggesting that immobilized metal affinity chromatography (IMAC) would be an effective primary purification method. A comprehensive purification strategy for this tagged protein might include:

• Immobilized Metal Affinity Chromatography (IMAC):

  • Using Ni-NTA or Co2+ resin to capture the His-tagged protein

  • Typical binding buffer: 20-50 mM Tris-HCl pH 8.0, 300-500 mM NaCl, 5-20 mM imidazole

  • Elution with increasing imidazole concentration (250-500 mM)

• Size Exclusion Chromatography (SEC):

  • For further purification and buffer exchange

  • Separates based on molecular size, useful for removing aggregates

• Ion Exchange Chromatography:

  • If needed for additional purity

  • Selection of cation or anion exchange based on protein pI

Depending on the specific experimental requirements, additional purification steps or tag removal might be necessary. The purification protocol should be optimized based on protein stability, yield requirements, and the intended downstream applications.

How can researchers verify the purity and integrity of recombinant DDB_G0284989?

To verify the purity and integrity of recombinant DDB_G0284989, researchers should employ multiple analytical techniques:

• SDS-PAGE Analysis:

  • The search results indicate that the protein has >90% purity as determined by SDS-PAGE

  • Expected molecular weight: ~8 kDa (71 amino acids) plus the weight of the His tag

  • Coomassie or silver staining for visualization

• Western Blot Analysis:

  • Using anti-His tag antibodies to confirm identity

  • Alternatively, specific antibodies against DDB_G0284989 if available

• Mass Spectrometry:

  • For exact mass determination and sequence verification

  • Techniques like MALDI-TOF or ESI-MS

• Size Exclusion Chromatography (SEC):

  • To assess homogeneity and detect aggregation

• Circular Dichroism (CD) Spectroscopy:

  • To evaluate secondary structure and proper folding

A typical purity assessment table might look like:

These methods collectively provide a comprehensive assessment of protein purity, identity, and structural integrity.

What is the predicted function of DDB_G0284989 based on sequence analysis?

To predict the function of DDB_G0284989, researchers should consider:

• Conducting computational analyses using tools like InterPro, Pfam, or SMART to identify conserved domains

• Performing homology modeling against structurally characterized proteins

• Analyzing the presence of signal peptides, transmembrane regions, or other functional motifs

• Investigating expression patterns across different developmental stages of Dictyostelium

Is DDB_G0284989 involved in any known cellular pathways in Dictyostelium?

To investigate potential pathway involvement, researchers could:

• Perform knockout or knockdown studies of DDB_G0284989 and analyze resulting phenotypes

• Conduct protein-protein interaction studies to identify binding partners

• Use phosphoproteomics to detect changes in signaling pathways when DDB_G0284989 is manipulated

• Analyze changes in gene expression patterns following DDB_G0284989 perturbation

Since Dictyostelium has been used to study various fundamental cellular processes including cell movement, chemotaxis, differentiation, and autophagy , examining DDB_G0284989's potential role in these processes would be worthwhile.

How does DDB_G0284989 expression change during the Dictyostelium life cycle?

To study expression patterns, researchers could:

• Perform RNA-seq or qRT-PCR analysis at different stages of the Dictyostelium life cycle:

  • Vegetative growth phase

  • Starvation response

  • Aggregation

  • Mound formation

  • Slug formation

  • Culmination and fruiting body formation

• Create reporter constructs (e.g., DDB_G0284989 promoter driving GFP expression) to visualize expression patterns in vivo during development

• Use Western blotting with specific antibodies to track protein levels throughout development

Understanding expression patterns could provide valuable clues about the protein's function, particularly if it shows stage-specific expression that correlates with specific developmental processes.

Are there any known phenotypes associated with DDB_G0284989 disruption?

To investigate phenotypes associated with DDB_G0284989 disruption, researchers could:

• Generate knockout mutants using CRISPR-Cas9 or other gene disruption methods

• Analyze mutant phenotypes across various aspects of Dictyostelium biology:

  • Growth rate in liquid culture and on bacterial lawns

  • Cell motility and chemotaxis

  • Response to various chemical compounds or environmental stressors

  • Developmental progression and morphology

  • Spore formation and viability

• Perform phenotypic rescue experiments by reintroducing the wild-type gene

This systematic phenotypic characterization could provide insights into the protein's biological role and identify potential functional redundancy with other genes.

How can CRISPR-based gene disruption be applied to study DDB_G0284989 function?

CRISPR-based gene disruption techniques have been successfully applied in Dictyostelium as mentioned in the search results , providing a powerful tool for studying gene function, including DDB_G0284989. While specific details about CRISPR application for DDB_G0284989 are not provided, a general methodological approach would include:

• Guide RNA (gRNA) Design:

  • Design gRNAs targeting exonic regions of DDB_G0284989, preferably early in the coding sequence

  • Use Dictyostelium-specific CRISPR design tools to optimize gRNA efficiency and minimize off-target effects

  • Consider designing multiple gRNAs to increase success rates

• CRISPR-Cas9 Delivery:

  • Construct a vector expressing Cas9 and the selected gRNA(s)

  • Include appropriate selection markers for Dictyostelium (e.g., G418 resistance)

  • Transform Dictyostelium cells using electroporation or other established methods

• Mutant Selection and Verification:

  • Select transformants using appropriate antibiotics

  • Verify gene disruption using PCR, sequencing, and Western blotting

  • Isolate clonal populations through serial dilution or cloning rings

• Phenotypic Analysis:

  • Compare growth, development, and behavior of mutants to wild-type cells

  • Conduct specific assays based on hypothesized function of DDB_G0284989

  • Create rescue strains by reintroducing the wild-type gene to confirm phenotype specificity

This approach would provide valuable insights into DDB_G0284989 function and its role in Dictyostelium biology.

What are the potential homologs of DDB_G0284989 in other species?

• Sequence-Based Homology Searches:

  • Use BLAST (Basic Local Alignment Search Tool) against comprehensive databases like UniProt, NCBI nr, and organism-specific databases

  • Employ position-specific iterative BLAST (PSI-BLAST) for detecting remote homologs

  • Use profile hidden Markov models (HMMs) with tools like HMMER for increased sensitivity

• Structural Prediction and Comparison:

  • Generate structural models using tools like AlphaFold2 or I-TASSER

  • Use structural alignment tools like DALI or TM-align to identify structural homologs

  • Analyze conserved structural features that might indicate functional similarity

• Phylogenetic Analysis:

  • Align sequences of potential homologs using tools like MUSCLE or MAFFT

  • Construct phylogenetic trees to visualize evolutionary relationships

  • Analyze patterns of sequence conservation across different taxonomic groups

The search results mention that another Dictyostelium protein, GrlJ, shares homology with an uncharacterized human protein, Q8NHA5 , suggesting that similar approaches could identify potential homologs of DDB_G0284989.

Could DDB_G0284989 be involved in bitter tastant detection like GrlJ?

Given that the search results identify GrlJ as a G-protein coupled receptor in Dictyostelium that regulates a phenylthiourea-dependent effect , it raises the possibility that other transmembrane proteins like DDB_G0284989 might have similar functions in detecting environmental compounds. To investigate whether DDB_G0284989 might be involved in bitter tastant detection, researchers could:

• Comparative Response Assays:

  • Expose wild-type, grlJ-, and DDB_G0284989- mutant cells to various bitter compounds (phenylthiourea, denatonium benzoate, quinine hydrochloride)

  • Analyze concentration-dependent cellular responses using motility assays as described for GrlJ

  • Construct a response profile table comparing the different cell lines:

  Compound	Concentration	Wild-type Response	grlJ- Response	DDB_G0284989- Response
  Phenylthiourea	2-5 mM	(baseline)	Partial resistance	?
  Denatonium benzoate	1-5 mM	(baseline)	?	?
  Quinine hydrochloride	0.5 mM	(baseline)	?	?

• Signal Transduction Analysis:

  • Investigate whether DDB_G0284989 interacts with G-proteins or affects PIP3 signaling pathways like GrlJ

  • Examine phosphorylation cascades and second messenger production in response to bitter compounds

  • Compare signaling events between wild-type and mutant cells

• Structure-Function Studies:

  • Analyze the transmembrane domain structure of DDB_G0284989 for potential ligand-binding sites

  • Create chimeric proteins with GrlJ to identify functional domains

  • Use site-directed mutagenesis to identify critical residues for tastant detection

This systematic approach would help determine whether DDB_G0284989 shares functional characteristics with GrlJ in bitter tastant detection.

How might DDB_G0284989 be related to cell motility or chemotaxis in Dictyostelium?

Dictyostelium is an established model for studying cell motility and chemotaxis , and the search results show that other transmembrane proteins like GrlJ affect cell movement in response to compounds like phenylthiourea . To investigate potential roles of DDB_G0284989 in cell motility or chemotaxis, researchers could:

• Baseline Motility Analysis:

  • Compare random cell movement parameters between wild-type and DDB_G0284989- cells using time-lapse microscopy

  • Track key motility metrics:

    • Cell velocity

    • Cell directionality

    • Cell shape changes (aspect ratio)

    • Pseudopod formation

    • Actin dynamics

• Chemotactic Response Assays:

  • Test directed migration toward various chemoattractants:

    • cAMP (primary Dictyostelium chemoattractant)

    • Folate (bacterial chemoattractant)

    • Other potential chemoattractants

• Gradient Sensing Mechanisms:

  • Analyze the spatial distribution of signaling components using fluorescent reporters

  • Investigate PIP3 dynamics, which are critical for chemotaxis and are affected by GrlJ

  • Examine cytoskeletal reorganization during gradient sensing

• Developmental Chemotaxis:

  • Assess the role of DDB_G0284989 during the aggregation phase of development

  • Analyze stream formation and mound morphology

  • Evaluate slug migration and phototactic/thermotactic responses

This comprehensive approach would help determine whether and how DDB_G0284989 contributes to the complex processes of cell motility and chemotaxis in Dictyostelium.

What techniques can be used to study the subcellular localization of DDB_G0284989?

Understanding the subcellular localization of DDB_G0284989 would provide important clues about its function. Based on the search results, Dictyostelium researchers have access to various expression constructs that enable studies on protein localization . To determine the subcellular localization of DDB_G0284989, researchers could employ:

• Fluorescent Protein Tagging:

  • Generate fusion constructs with fluorescent proteins (GFP, RFP, etc.)

  • Create both N- and C-terminal fusions to account for potential interference with localization signals

  • Express under native or inducible promoters

  • Visualize using confocal or super-resolution microscopy

• Co-localization Studies:

  • Perform dual-labeling experiments with known organelle markers:

    • Plasma membrane: FM4-64 or membrane-targeted fluorescent proteins

    • Endoplasmic reticulum: ER-Tracker or calreticulin-GFP

    • Golgi apparatus: golvesin-GFP

    • Endosomes/lysosomes: RFP-Rab7 or lysotracker

    • Mitochondria: MitoTracker or mitochondrial-targeted GFP

• Immunofluorescence:

  • Develop specific antibodies against DDB_G0284989

  • Use anti-tag antibodies if working with tagged versions

  • Optimize fixation and permeabilization protocols for transmembrane proteins

• Subcellular Fractionation:

  • Separate cellular components through differential centrifugation

  • Analyze protein distribution across fractions using Western blotting

  • Create a quantitative distribution profile across cellular compartments

A systematic application of these techniques would provide a comprehensive understanding of where DDB_G0284989 functions within the cell, offering important insights into its biological role.