Recombinant Danio rerio UPF0694 transmembrane protein C14orf109 homolog (si:dkeyp-55f12.4, zgc:112233)

What is the basic structural characterization of UPF0694 transmembrane protein C14orf109 homolog?

The UPF0694 transmembrane protein C14orf109 homolog in Danio rerio is a transmembrane protein with 142 amino acids. Its full amino acid sequence is: MMNFRQRMGWIGVGLYLLASVAAVYYIFEISQTYNRLALAQVEKTSGAQAKWPGDASSSSSPSSTSWIVTLKTRLLLLPFWVWATIFLLPYLQVFLFLYSCTRADPKTVGYCILPICLAVLCNRHQTFTKASNQISRLQLIDT . Structurally, this protein belongs to a classification of transmembrane proteins that span the lipid bilayer. Based on common methodologies for transmembrane protein analysis, researchers can predict its topology using computational tools like PSIPRED or TMHMM to identify transmembrane helix regions, similar to approaches used for other proteins such as MC1R in zebrafish .

What are the recommended storage and handling protocols for recombinant Danio rerio UPF0694 protein?

For optimal preservation of protein integrity and activity, store the recombinant UPF0694 protein at -20°C for regular use, and at -20°C or -80°C for extended storage periods. The protein is typically supplied in a Tris-based buffer with 50% glycerol, which has been optimized for stability . To minimize protein degradation, avoid repeated freeze-thaw cycles, as this can compromise structural integrity and biological activity. When working with the protein, prepare working aliquots and store them at 4°C for up to one week to avoid repeated freeze-thaw cycles of the stock solution . Before experimental use, thaw aliquots on ice and centrifuge briefly to collect contents at the bottom of the tube.

How can researchers verify the identity and purity of the recombinant UPF0694 protein?

Methodological approach for identity and purity verification includes:

• SDS-PAGE: Run the protein on a 10-15% gel alongside appropriate molecular weight markers to confirm the expected size of approximately 142 amino acids.

• Western Blot: Utilize antibodies specific to the protein or any tags incorporated during recombinant production.

• Mass Spectrometry: Employ LC-MS/MS for definitive identification of the protein through peptide mass fingerprinting.

• Circular Dichroism: Assess the secondary structure composition to ensure proper folding.

• Size Exclusion Chromatography: Evaluate protein homogeneity and absence of aggregation.

Researchers should establish acceptance criteria for each method based on the specific experimental requirements, typically setting purity thresholds of >90% for functional studies and >95% for structural analyses.

What expression systems are most effective for producing recombinant Danio rerio UPF0694 protein?

For recombinant expression of the UPF0694 transmembrane protein, researchers should consider several expression systems, each with distinct advantages:

For the UPF0694 transmembrane protein, insect cell expression may provide the best balance between proper folding of transmembrane domains and reasonable yield. Codon optimization for the expression system of choice is critical for efficient expression, as is the inclusion of appropriate affinity tags (positioned to avoid interference with transmembrane domains) for purification.

What methodologies are most effective for studying protein-protein interactions involving the UPF0694 transmembrane protein?

When investigating protein-protein interactions of the UPF0694 transmembrane protein, researchers should employ complementary approaches:

• Co-immunoprecipitation (Co-IP): Use antibodies against UPF0694 or potential interacting partners to pull down protein complexes from zebrafish cell lysates, followed by Western blot or mass spectrometry.

• Proximity-based labeling: Employ BioID or APEX2 fusion proteins to identify proximal proteins in the native cellular environment.

• Yeast two-hybrid membrane system (MYTH): Specifically adapted for membrane proteins, this system can detect interactions between the UPF0694 transmembrane protein and other proteins.

• Fluorescence resonance energy transfer (FRET): Tag UPF0694 and candidate interacting proteins with appropriate fluorophore pairs to detect interactions in living cells.

• Surface plasmon resonance (SPR): Measure binding kinetics and affinities between purified UPF0694 and candidate partners.

For transmembrane proteins like UPF0694, detergent selection is critical during extraction and purification to maintain native conformation. Consider using mild detergents such as DDM, LMNG, or digitonin, which effectively solubilize membrane proteins while preserving structural integrity.

How can homology modeling be applied to predict the structure of UPF0694 transmembrane protein?

Homology modeling provides a valuable approach for predicting the three-dimensional structure of UPF0694 transmembrane protein. Following a methodology similar to that used for other zebrafish proteins , researchers should:

The resulting model can provide insights into the protein's functional domains, potential binding sites, and structural characteristics, guiding further experimental design.

What are the challenges in studying transmembrane topology of UPF0694 protein and how can they be addressed?

Determining the transmembrane topology of UPF0694 presents several methodological challenges:

• Experimental Difficulties:

  • Resistance to crystallization

  • Instability in detergent solutions

  • Aggregation during purification

• Methodological Solutions:

  • Utilize cysteine scanning mutagenesis with membrane-impermeable sulfhydryl reagents to map exposed regions

  • Apply protease protection assays to identify cytoplasmic versus extracellular domains

  • Employ fluorescence protease protection (FPP) assay using GFP-tagged constructs at N- and C-termini

  • Implement glycosylation mapping by introducing N-glycosylation sites at various positions

• Computational Approaches:

  • Apply multiple prediction algorithms (TMHMM, Phobius, TOPCONS) and identify consensus predictions

  • Validate in silico predictions with limited experimental data points

  • Use evolutionary conservation analysis to identify functionally important regions

A combined approach using both experimental and computational methods yields the most reliable topology model, which is essential for functional studies and targeting specific domains for interaction analysis.

How can molecular dynamics simulations enhance our understanding of UPF0694 protein function?

Molecular dynamics (MD) simulations provide valuable insights into the dynamic behavior and functional mechanisms of UPF0694 transmembrane protein. Following methodology similar to that applied to other zebrafish membrane proteins :

MD simulations can reveal conformational changes, identify potential binding pockets, and elucidate the effects of mutations on protein stability and function. For UPF0694, particular attention should be paid to the transmembrane regions and any conserved motifs that might be functionally significant.

What is the evolutionary conservation pattern of UPF0694 protein across species and what does it suggest about function?

Evolutionary conservation analysis of UPF0694 protein provides critical insights into functionally important regions. Methodological approach:

• Multiple Sequence Alignment (MSA):

  • Collect UPF0694 homologs from diverse species using BLAST against UniProt

  • Perform MSA using MUSCLE or CLUSTALW algorithms

  • Visualize conservation patterns using tools like Jalview or WebLogo

• Phylogenetic Analysis:

  • Construct phylogenetic trees using Maximum Likelihood or Bayesian methods

  • Calculate evolutionary rates using PAML to identify sites under purifying or positive selection

  • Compare with other UPF family proteins to determine unique features

• Domain Conservation Assessment:

  • Map conservation scores onto the structural model

  • Identify highly conserved motifs that likely correspond to functional sites

  • Compare transmembrane domains with cytoplasmic/extracellular regions

• Functional Prediction:

  • Correlate conservation patterns with known functional domains of related proteins

  • Identify putative interaction interfaces based on surface conservation

What are the established and potential functions of UPF0694 transmembrane protein in zebrafish development?

The UPF0694 transmembrane protein's role in zebrafish development remains an active area of investigation. Current methodological approaches to elucidate its function include:

• Spatiotemporal Expression Analysis:

  • Perform in situ hybridization to map gene expression patterns during different developmental stages

  • Use RT-qPCR to quantify expression levels across tissues and developmental timepoints

  • Employ fluorescent reporter constructs to track expression in real-time

• Loss-of-Function Studies:

  • Generate knockout models using CRISPR/Cas9 targeting si:dkeyp-55f12.4 gene

  • Apply morpholino knockdown for transient functional analysis

  • Analyze resulting phenotypes for developmental abnormalities

• Gain-of-Function Analysis:

  • Overexpress the protein using mRNA injection at one-cell stage

  • Create transgenic lines with inducible expression

  • Assess developmental consequences of altered expression levels

• Proteomic Interaction Studies:

  • Perform pull-down assays coupled with mass spectrometry to identify interacting partners

  • Validate interactions using co-immunoprecipitation and proximity ligation assays

While definitive functions remain to be established, comparison with related proteins suggests potential roles in membrane organization, signaling pathways, or protein trafficking. Understanding its function requires integrating data from these multiple experimental approaches and correlating findings with known developmental processes.

How can CRISPR/Cas9 technology be optimized for functional studies of UPF0694 transmembrane protein in zebrafish?

CRISPR/Cas9-mediated genome editing offers powerful approaches for functional characterization of UPF0694 transmembrane protein. Optimization strategies include:

• gRNA Design and Validation:

  • Target conserved exons encoding transmembrane domains or other predicted functional regions

  • Design multiple gRNAs (3-4) targeting different regions of the si:dkeyp-55f12.4 gene

  • Test gRNA efficiency using in vitro cleavage assays before zebrafish experiments

  • Prioritize gRNAs with minimal predicted off-target effects using tools like CHOPCHOP or CRISPOR

• Delivery Methods:

  Method	Advantages	Limitations	Recommended Application
  Microinjection	Established technique, reliable	Limited to early embryos	Standard knockout generation
  Electroporation	Higher efficiency, can target specific tissues	More tissue damage	Tissue-specific studies
  Lipofection	Less invasive	Lower efficiency	Alternative when microinjection fails

• Knockout Verification Strategies:

  • T7 Endonuclease I assay for initial mutation detection

  • High-resolution melt analysis for rapid screening

  • Sanger sequencing for precise mutation characterization

  • Western blotting to confirm protein loss or truncation

• Phenotypic Analysis Pipeline:

  • Morphological assessment at key developmental stages

  • Behavioral testing to identify subtle phenotypes

  • Histological analysis of affected tissues

  • Molecular profiling (RNA-seq) to identify downstream effects

• Advanced Genome Editing Approaches:

  • Base editing for introducing specific point mutations

  • Prime editing for precise sequence modifications

  • Conditional knockout systems (e.g., Cre-loxP) for temporal control

For transmembrane proteins like UPF0694, consider generating domain-specific mutations rather than complete knockouts to dissect the function of different protein regions.

What signaling pathways might UPF0694 transmembrane protein participate in, and how can this be investigated?

Investigating the potential involvement of UPF0694 transmembrane protein in signaling pathways requires a systematic approach:

• Bioinformatic Prediction:

  • Analyze the protein sequence for conserved signaling motifs (phosphorylation sites, interaction domains)

  • Perform network analysis using tools like STRING to identify potential functional associations

  • Compare with known interactors of homologous proteins in other species

• Phosphoproteomic Analysis:

  • Immunoprecipitate UPF0694 and analyze post-translational modifications

  • Investigate changes in phosphorylation status under different stimuli

  • Map identified phosphorylation sites to potential kinase recognition motifs

• Pathway Perturbation Experiments:

  • Treat zebrafish embryos or cells with pathway-specific inhibitors and assess UPF0694 expression/localization

  • Monitor downstream effects of UPF0694 knockdown on known pathway components

  • Perform rescue experiments with activated pathway components

• Transcriptomic Profiling:

  • Compare gene expression profiles between wild-type and UPF0694-deficient zebrafish

  • Apply pathway enrichment analysis to identify significantly affected signaling networks

  • Validate key differentially expressed genes using qRT-PCR

• Protein-Protein Interaction Mapping:

  • Perform BioID or APEX2 proximity labeling to identify proteins in close proximity to UPF0694

  • Validate interactions using co-immunoprecipitation and FRET analysis

  • Construct interaction networks to identify potential signaling hubs

Based on its transmembrane nature, UPF0694 might participate in receptor-mediated signaling, membrane trafficking, or organelle communication pathways. Special attention should be given to analyzing its potential interactions with G-protein coupled receptors or other signaling complexes in the membrane environment.

How can researchers overcome solubility challenges when working with recombinant UPF0694 transmembrane protein?

Solubilizing transmembrane proteins like UPF0694 for biochemical and structural studies presents significant challenges. Methodological solutions include:

• Detergent Screening Protocol:

  • Systematically test multiple detergent classes:

    • Mild (DDM, LMNG, digitonin)

    • Intermediate (DM, UDM)

    • Harsh (LDAO, OG)

  • Assess protein stability in each detergent using size exclusion chromatography

  • Optimize detergent concentration through thermal stability assays

• Solubilization Strategy Matrix:

  Approach	Methodology	Advantages	Applications
  Detergent micelles	Standard extraction with optimized detergent	Well-established	Biochemical assays
  Amphipols	Detergent exchange to A8-35 or PMAL	Enhanced stability	Structural studies
  Nanodiscs	Reconstitution with MSP proteins and lipids	Native-like environment	Functional studies
  SMALPs	Direct extraction with SMA copolymers	Preserves annular lipids	Native mass spec
  Saposin lipoprotein nanoparticles	Reconstitution with saposin A	Small particle size	Cryo-EM studies

• Protein Engineering Approaches:

  • Truncate flexible termini that may cause aggregation

  • Introduce solubility-enhancing mutations in exposed residues

  • Create fusion constructs with soluble protein partners

  • Add thermostabilizing mutations identified through computational prediction

• Co-expression Strategies:

  • Express with natural binding partners to enhance folding and stability

  • Co-express with membrane-protein-specific chaperones

• Buffer Optimization:

  • Screen pH ranges (typically 6.5-8.0)

  • Test various salt concentrations (100-500 mM)

  • Include glycerol (5-10%) as a stabilizing agent

  • Add specific lipids that may be required for stability

These approaches should be tested systematically, evaluating protein activity and homogeneity at each step to ensure that the solubilized UPF0694 remains in its native conformation.

What techniques are most appropriate for studying the subcellular localization of UPF0694 protein in zebrafish cells?

Determining the subcellular localization of UPF0694 protein requires complementary imaging techniques:

• Fluorescent Protein Fusion Approach:

  • Generate C- and N-terminal GFP/mCherry fusions of UPF0694

  • Express in zebrafish cell lines or embryos using appropriate promoters

  • Assess impact of tags on protein function through rescue experiments

  • Perform live imaging to track dynamic localization patterns

• Immunofluorescence Protocol:

  • Develop specific antibodies against UPF0694 or use commercial options if available

  • Optimize fixation methods (4% PFA for general structures, methanol for membrane proteins)

  • Perform co-localization studies with organelle markers:

    • Plasma membrane: Na+/K+ ATPase, WGA

    • ER: Calnexin, KDEL-tagged proteins

    • Golgi: GM130, TGN46

    • Endosomes: Rab5, Rab7, Rab11

    • Lysosomes: LAMP1, LAMP2

• High-Resolution Imaging Techniques:

  • Super-resolution microscopy (STED, PALM, STORM) for detailed localization beyond diffraction limit

  • Correlative light and electron microscopy (CLEM) to combine fluorescence with ultrastructural context

  • Expansion microscopy for physical magnification of subcellular structures

• Biochemical Fractionation:

  • Separate cellular components through differential centrifugation

  • Perform Western blotting of fractions to detect UPF0694

  • Compare distribution with known organelle markers

• Proximity Labeling:

  • Fuse UPF0694 with BioID or APEX2

  • Identify proximal proteins through streptavidin pulldown and mass spectrometry

  • Map the protein's microenvironment within cellular compartments

For transmembrane proteins like UPF0694, particular attention should be paid to membrane trafficking pathways, as the protein may dynamically shuttle between compartments in response to cellular signals.

How can researchers design effective experiments to study the structure-function relationship of UPF0694 transmembrane protein?

Investigating structure-function relationships of UPF0694 requires systematic experimental design:

• Domain Mapping Strategy:

  • Generate truncation constructs removing specific domains

  • Create chimeric proteins by swapping domains with related proteins

  • Assess functional consequences using established assays

  • Correlate functional changes with structural elements

• Site-Directed Mutagenesis Approach:

  • Identify conserved residues through sequence alignment

  • Target transmembrane regions and potential functional motifs

  • Create single and multiple point mutations

  • Employ alanine-scanning for systematic functional mapping

• Experimental Matrix for Functional Assessment:

  Mutation Type	Target Residues	Expected Impact	Assay Methods
  Conservative	Hydrophobic core residues	Mild structural changes	Thermal stability, activity assays
  Non-conservative	Charged/polar residues	Significant functional impact	Binding studies, localization
  Cysteine substitution	Surface-exposed positions	Minimal disruption, allows labeling	Accessibility studies, FRET
  Deletion	Non-essential loops	Variable based on region	Expression level, trafficking

• Structure Validation Techniques:

  • Circular dichroism to assess secondary structure changes

  • Limited proteolysis to probe conformational alterations

  • Thermal shift assays to measure stability differences

  • Molecular dynamics simulations to predict structural impacts of mutations

• Correlation Analysis:

  • Map experimental results to structural models

  • Identify critical residues/motifs for specific functions

  • Develop predictive models of structure-function relationships

This systematic approach enables researchers to build a comprehensive map of how specific structural elements of UPF0694 contribute to its biological functions, guiding future studies on regulation and potential therapeutic targeting.