Recombinant Protein SEY1 homolog 2 (TVAG_100140)

What are the optimal storage and handling conditions for Recombinant SEY1 homolog 2?

For optimal preservation of protein activity and stability, Recombinant SEY1 homolog 2 requires specific storage and handling protocols:

Storage Recommendations:

• Store at -20°C/-80°C upon receipt

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

• Avoid repeated freeze-thaw cycles as this significantly reduces protein activity

Buffer Composition:

• Typically supplied in Tris/PBS-based buffer with 6% Trehalose, pH 8.0

• For long-term storage, addition of glycerol (final concentration 50%) is recommended

Reconstitution Protocol:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol (5-50% final concentration) and prepare working aliquots

• Flash freeze aliquots in liquid nitrogen before storing at -80°C for maximum stability

Quality Control Metrics:

• Purity: Should be >90% as determined by SDS-PAGE

• Activity: Functional assays should be performed after reconstitution to confirm GTPase activity is preserved

What experimental designs are recommended for initial characterization of SEY1 homolog 2?

When designing experiments for initial characterization of SEY1 homolog 2, researchers should employ a systematic approach:

Pre-Experimental Planning:

• Clearly define research questions and formulate testable hypotheses

• Identify independent variables (e.g., protein concentration, nucleotide conditions) and dependent variables (e.g., GTPase activity, dimerization rate)

• Control for extraneous variables (e.g., buffer conditions, temperature)

Recommended Experimental Designs:

A basic characterization workflow should include:

• Biochemical Characterization:

  • GTPase activity assays with varying nucleotide concentrations

  • Size exclusion chromatography to assess oligomeric states

  • Thermal stability assays (DSF/DSC) with different nucleotides

• True Experimental Design Approach:

  • Establish control groups and experimental groups

  • Randomly assign samples to treatment conditions

  • Manipulate independent variables systematically

• Random Distribution of Variables:

  • Ensure sufficient technical and biological replicates

  • Randomize the order of experiments to prevent systematic errors

Data Analysis Considerations:

• Apply appropriate statistical methods based on data distribution

• Consider using factorial designs to examine interactions between variables

• Document all experimental conditions thoroughly for reproducibility

How do the crystal structures of SEY1 homolog 2 provide insight into its mechanism of action?

The crystal structures of SEY1 homolog 2 reveal crucial information about its conformational changes and functional mechanisms:

Structural Data Analysis:
The N-terminal cytosolic domain of Sey1p has been crystallized in different nucleotide-bound states, yielding structures with the following parameters:

Table adapted from crystallographic data

Mechanistic Insights:
The structural analysis reveals that:

• Nucleotide-Dependent Dimerization:

  • In the GDP/AlF₄⁻-bound state (mimicking the transition state of GTP hydrolysis), Sey1p forms a dimer with GTPase domains facing each other

  • The GDP-bound form shows a different conformation with the stalk domain associated with the GTPase domain of the same molecule

• Conformational Changes:

  • The linker regions of two Sey1p molecules cross one another in the dimer

  • This allows close association of the tops of the stalk domains

  • The stalk domain is composed of four 3HBs, with each 3HB connected to the next

• Functional Implications:

  • The structural rearrangements suggest a mechanism for membrane tethering and fusion

  • Nucleotide binding and hydrolysis drive conformational changes that bring membranes into close proximity

Experimental Approaches for Further Analysis:

• Hydrogen-deuterium exchange mass spectrometry to map conformational dynamics

• Site-directed mutagenesis of key residues at interaction interfaces

• Molecular dynamics simulations to model transitions between different nucleotide states

What are the most effective methodologies for studying SEY1 homolog 2's role in membrane fusion?

To effectively investigate SEY1 homolog 2's role in membrane fusion, researchers should employ complementary in vitro and cellular approaches:

In Vitro Reconstitution Assays:

• Liposome Fusion Assays:

  • Reconstitute purified SEY1 homolog 2 into liposomes with defined lipid compositions

  • Monitor fusion using FRET-based lipid mixing assays

  • Quantify both lipid mixing (hemifusion) and content mixing (complete fusion)

  • Include controls with GTPase-deficient mutants (e.g., K50A P-loop mutant)

• GTPase Activity Coupling:

  • Correlate GTPase activity with fusion efficiency using real-time assays

  • Test the effects of nucleotide analogs and transition state mimics

  • Investigate the kinetics of fusion relative to GTP hydrolysis cycles

Cellular Assays:

• Rescue Experiments in Mammalian Cells:
  Studies have shown that Sey1p can replace ATLs (atlastins) in mammalian cells when ATLs are knocked down. Key methodology includes:

  • siRNA-mediated depletion of endogenous ATLs (ATL2 and ATL3)

  • Visualization of ER morphology using calreticulin as a marker

  • Expression of wild-type or mutant Sey1p to assess rescue efficiency

  • Quantification of ER network restoration

• Structure-Function Analysis:
  When testing SEY1 function in cells, critical mutants should include:

  • K50A (P-loop mutant)

  • L233A (dimer interface mutant)

  • L297A, V298A (GTPase–stalk interface mutants)

  • Δ1/2stalk (stalk deletion mutant)

Quantitative Analysis Approaches:

• Develop scoring systems for ER morphology (normal vs. unbranched)

• Use high-content imaging to analyze large numbers of cells

• Apply machine learning algorithms for unbiased morphological classification

• Compare expression levels of wild-type and mutant proteins using Western blotting

How can quasi-experimental designs be applied to study SEY1 homolog 2 in complex biological systems?

When studying SEY1 homolog 2 in complex biological systems where full experimental control is not possible, quasi-experimental designs offer valuable alternatives:

Quasi-Experimental Approaches:

• Non-Equivalent Control Group Designs:

  • Compare cell lines with different endogenous levels of SEY1 expression

  • Use CRISPR-Cas9 to generate knockout or knockin cell lines

  • Apply statistical controls to account for pre-existing differences between groups

• Interrupted Time Series Analysis:

  • Monitor cellular processes before and after acute inhibition of SEY1 function

  • Use small molecule inhibitors or optogenetic tools for temporal control

  • Collect multiple data points before and after intervention

  • Apply segmented regression analysis to identify change points

• Stepped Wedge Design for Multi-Condition Testing:

  • Sequentially introduce SEY1 mutations or inhibitors across different cell populations

  • Each group serves as its own control over time

  • Particularly useful for studying SEY1's role in development or differentiation processes

Statistical Considerations:

Ethical and Practical Advantages:

• Allows research in primary tissues where random assignment is not possible

• Enables longitudinal studies of SEY1 function in development or disease

• Provides framework for translational research bridging basic and clinical studies

What experimental controls are essential when studying GTPase activity of SEY1 homolog 2?

Rigorous experimental controls are crucial for accurate characterization of SEY1 homolog 2's GTPase activity:

Essential Controls for GTPase Assays:

• Negative Controls:

  • Buffer-only reactions (no protein)

  • Catalytically inactive mutants (K50A mutation in P-loop)

  • Heat-denatured protein samples

  • GTPase reactions in the absence of essential cofactors (e.g., Mg²⁺)

• Positive Controls:

  • Well-characterized GTPase with known kinetic parameters

  • Previously validated batches of SEY1 homolog 2

  • Reactions with known GTPase activators

• Specificity Controls:

  • Test multiple nucleotides (GTP, ATP, CTP, etc.)

  • Include non-hydrolyzable analogs (GMP-PNP, GTPγS)

  • Transition state analogs (GDP/AlF₄⁻)

Critical Experimental Variables to Control:

• Reaction Conditions:

  • Temperature (typically 25°C or 37°C)

  • pH (optimal range 7.0-8.0)

  • Ionic strength (150 mM NaCl standard)

  • Divalent cation concentration (typically 1-5 mM MgCl₂)

  • Reducing agents (DTT or TCEP concentration)

• Protein-Specific Factors:

  • Protein concentration determined by validated methods

  • Batch-to-batch consistency verification

  • Storage duration and freeze-thaw cycles documented

  • Oligomeric state verification before assays

Dose-Response Experimental Design:

• Test multiple protein concentrations to confirm linearity

• Vary substrate (GTP) concentration to determine Km and Vmax

• Consider time-course experiments to establish initial rates

• Use Michaelis-Menten or allosteric kinetic models as appropriate

Data Validation Approaches:

• Use multiple detection methods (e.g., colorimetric, HPLC, coupled enzyme assays)

• Include internal standards for quantification

• Perform statistical analysis to identify outliers

• Report all assay conditions and controls in publications

How can structure-function relationships in SEY1 homolog 2 be investigated through mutagenesis?

Strategic mutagenesis approaches provide powerful tools for dissecting structure-function relationships in SEY1 homolog 2:

Mutagenesis Strategy Design:

• Target Domain Selection:
  Based on crystal structures, key domains for mutagenesis include:

  • GTPase domain (focus on nucleotide binding and hydrolysis sites)

  • Dimer interface residues

  • GTPase-stalk interface

  • Membrane interaction regions

• Types of Mutations to Consider:

  • Alanine scanning of conserved motifs

  • Conservative vs. non-conservative substitutions

  • Domain swaps with related GTPases

  • Deletion of specific structural elements (e.g., Δ1/2stalk)

Functional Analysis Framework:

• Biochemical Characterization:
  For each mutant, analyze:

  • GTPase activity (kcat and Km)

  • Nucleotide binding affinity

  • Dimerization properties

  • Thermal stability changes

  • Comparative analysis with wild-type protein

• Cellular Function Assessment:

  • Rescue experiments in cells lacking endogenous SEY1/ATLs

  • Quantitative scoring of ER morphology (normal vs. unbranched)

  • Colocalization with ER markers

  • Dynamics analysis using fluorescence recovery after photobleaching (FRAP)

Critical Mutations Based on Published Structures:

Table based on functional data from structural studies

Advanced Mutagenesis Approaches:

• Use evolutionary conservation analysis to identify functionally important residues

• Apply deep mutational scanning for comprehensive structure-function mapping

• Consider conditional mutants (temperature-sensitive) for temporal control

• Use chimeric constructs between SEY1 homologs to identify species-specific functions

What are the best practices for developing robust experimental designs to study SEY1 and membrane dynamics?

Developing robust experimental designs for studying SEY1 and membrane dynamics requires careful consideration of multiple factors:

Experimental Design Framework:

• Define Clear Research Questions:

  • Formulate specific, testable hypotheses about SEY1 function

  • Establish measurable outcomes for membrane dynamics

  • Identify potential confounding variables

• Variable Selection and Control:

  • Independent variables: SEY1 expression levels, mutations, nucleotide conditions

  • Dependent variables: membrane fusion rates, morphological changes, protein interactions

  • Control variables: temperature, pH, membrane composition

• Randomization and Blinding:

  • Randomly assign samples to treatment groups

  • Use blinded analysis for subjective measurements (e.g., morphology scoring)

  • Include multiple observers for validation

Advanced Design Strategies:

• Factorial Designs:

  • Test multiple variables simultaneously (e.g., protein concentration × nucleotide type × membrane composition)

  • Analyze interaction effects between variables

  • Optimize experimental conditions efficiently

• Time-Series Analyses:

  • Track membrane dynamics over time after SEY1 activation

  • Establish baseline measurements before intervention

  • Use appropriate statistical methods for repeated measures

• Cross-Validation Approaches:

  • Verify findings using multiple experimental systems

  • Combine in vitro reconstitution with cellular assays

  • Apply different measurement techniques for the same phenomenon

Methodological Controls and Considerations:

• System-Specific Controls:

  • For in vitro systems: protein-free liposomes, GTPase-dead mutants

  • For cellular systems: untransfected cells, irrelevant protein overexpression

  • For imaging: fluorophore-only controls, photobleaching corrections

• Statistical Power and Sample Size:

  • Perform power analysis to determine appropriate sample sizes

  • Consider effect size from preliminary data or literature

  • Plan for multiple experimental replicates (biological and technical)

• Data Validation Strategy:

  • Establish quantitative criteria for data inclusion/exclusion

  • Use multiple analytical methods when possible

  • Apply appropriate statistical tests based on data distribution

Documentation and Reporting:

• Preregister experimental designs when possible

• Document all experimental conditions thoroughly

• Report all results, including negative or inconclusive findings

• Share detailed protocols to enhance reproducibility

How can recombinant SEY1 homolog 2 be used to investigate pathogen-host interactions in Trichomonas vaginalis infections?

Recombinant SEY1 homolog 2 offers unique opportunities to study Trichomonas vaginalis pathogenesis and host-pathogen interactions:

Research Applications:

• Structural Vaccinology Approach:

  • Use purified recombinant SEY1 homolog 2 to identify surface-exposed epitopes

  • Design subunit vaccine candidates targeting conserved regions

  • Test immunogenicity in animal models

  • Evaluate protective efficacy against challenge infections

• Drug Target Validation:

  • Develop high-throughput screening assays using purified protein

  • Identify small molecule inhibitors of SEY1 GTPase activity

  • Test compound specificity against human homologs

  • Evaluate the impact of SEY1 inhibition on T. vaginalis viability

Experimental Design Considerations:

• Comparative Studies with Host Proteins:

  • Express and purify human homologs alongside T. vaginalis SEY1

  • Compare biochemical properties and structure

  • Identify parasite-specific features for selective targeting

  • Use chimeric proteins to map functional differences

• Host-Pathogen Interaction Studies:

  • Investigate SEY1's role in T. vaginalis adaptation to host environment

  • Study membrane dynamics during host cell attachment

  • Examine SEY1 regulation during different infection stages

  • Develop cell-based assays modeling key aspects of infection

Advanced Methodological Approaches:

• Protein Localization and Trafficking:

  • Generate antibodies against recombinant SEY1 for immunolocalization

  • Create fluorescently tagged versions for live-cell imaging

  • Track dynamics during host cell interaction

  • Correlate localization with infection stages

• Protein-Protein Interaction Analysis:

  • Use recombinant SEY1 as bait in pull-down assays

  • Identify host cell targets using proximity labeling approaches

  • Confirm interactions through complementary methods (co-IP, FRET)

  • Map interaction domains through mutagenesis

• Experimental Controls for Pathogenesis Studies:

  • Include non-pathogenic trichomonad species as controls

  • Use GTPase-deficient mutants to assess enzyme activity requirements

  • Compare wild-type and SEY1-depleted parasites in infection models

  • Evaluate host cell responses to purified SEY1 vs. whole parasites

Translational Research Design:

• Develop SEY1-based diagnostic assays for T. vaginalis detection

• Screen for immunodominant epitopes in patient samples

• Identify correlates of protection in naturally resistant individuals

• Design structure-based inhibitors with therapeutic potential