Recombinant Mouse Glutamyl-tRNA (Gln) amidotransferase subunit C, mitochondrial (Gatc)

What is the structural organization of recombinant mouse Gatc protein?

Recombinant mouse Gatc is a secreted protein with a size of approximately 17.5 kDa, though it may appear larger on SDS-PAGE due to post-translational modifications . The protein contains a characteristic cysteine-rich region that is important for its function in the GatCAB complex.

When expressed recombinantly, mouse Gatc typically comprises residues Cys25-Phe222, often with an additional C-terminal tag (such as 10-His) for purification purposes . The protein features specific domains including:

• An N-terminal signal peptide (residues 1-24)

• A functional domain involved in the transamidation reaction

• Regions that facilitate interaction with the other GatCAB complex subunits

The structural characteristics of Gatc enable it to participate effectively in the transamidation process, ensuring proper mitochondrial translation .

How does dysfunction of Gatc affect mitochondrial protein synthesis?

Interference with Gatc expression or function in mouse cells produces significant defects in mitochondrial translation without affecting the stability of newly synthesized proteins . The consequences of Gatc dysfunction include:

• Impairment of the oxidative phosphorylation system

• Significant increase in reactive oxygen species (ROS) levels

• Disruption of mitochondrial energy production

Mass spectrometry analysis of mitochondrial proteins from cells with impaired Gatc function reveals no glutamic acid found in positions normally occupied by glutamine. This suggests that misaminoacylated Glu-tRNA(Gln) is rejected from the translational apparatus, maintaining the fidelity of mitochondrial protein synthesis despite the disruption in the transamidation pathway .

In humans, mutations in GATC gene are associated with Combined Oxidative Phosphorylation Deficiency 42, a mitochondrial disease characterized by impaired energy production .

What experimental approaches are recommended for studying Gatc function in mouse models?

When investigating Gatc function in mouse models, researchers should consider multiple experimental approaches to comprehensively understand its role in mitochondrial translation:

Genetic Manipulation Strategies

Functional Assays

To assess the impact of Gatc manipulation on mitochondrial function, researchers should measure:

• Mitochondrial protein synthesis rates using 35S-methionine pulse labeling

• Oxygen consumption rate (OCR) to evaluate oxidative phosphorylation efficiency

• ROS production using fluorescent indicators

• ATP synthesis capacity in isolated mitochondria

• Assembly of respiratory chain complexes via Blue Native PAGE

For comprehensive analysis, combine these approaches with proteomics to identify potential compensatory mechanisms that may activate when Gatc function is compromised.

How should researchers design experiments to evaluate recombinant Gatc enzyme activity in vitro?

When designing experiments to evaluate recombinant Gatc enzyme activity, researchers should consider the following methodological framework:

Protein Expression and Purification

• Express recombinant mouse Gatc in E. coli as a single, non-glycosylated polypeptide chain

• Include a His-tag for purification by proprietary chromatographic techniques

• Reconstitute the lyophilized protein at 100 μg/mL in sterile PBS containing at least 0.1% human or bovine serum albumin

• For prolonged storage, dilute to working aliquots in a 0.1% BSA solution, store at -80°C and avoid repeated freeze-thaw cycles

Assay Design for Enzyme Activity

When designing experiments to test Gatc enzyme activity, follow these experimental guidelines:

• Choose appropriate independent variables:

  • Temperature (use water baths to minimize fluctuations)

  • pH (test range 5.0-9.0)

  • Substrate concentration (misacylated Glu-tRNA(Gln))

  • Presence of inhibitors

  • ATP concentration

• Control extraneous variables:

  • Ensure consistent co-factor concentrations (ATP, glutamine)

  • Maintain consistent buffer conditions

  • Control for enzyme concentration across experimental conditions

  • Include negative controls (no enzyme) and positive controls (known active enzyme)

• Measure activity using:

  • Rate of ATP hydrolysis (coupled enzyme assay)

  • Formation of correctly charged Gln-tRNA(Gln) (measured by specific aminoacylation assays)

  • Thin-layer chromatography to separate and quantify reaction products

The resulting data should be analyzed for enzyme kinetics parameters including Km, Vmax, and catalytic efficiency (kcat/Km).

What strategies are effective for investigating the interactions between Gatc and other components of the GatCAB complex?

Understanding the interactions between Gatc and other components of the GatCAB complex requires a multi-faceted approach:

Structural Analysis Techniques

Biochemical Interaction Analysis

• Co-immunoprecipitation assays: Use antibodies against Gatc to pull down the entire GatCAB complex from mitochondrial extracts

• Pull-down assays: Use recombinant tagged Gatc to identify interaction partners

• Surface plasmon resonance: Measure binding kinetics between Gatc and other subunits

• Isothermal titration calorimetry: Determine thermodynamic parameters of binding

Genetic Approaches

Implement genetic crossing studies in a GATC genetic algorithm framework to identify potential genetic interactions. This approach combines elements of selection, crossover, and mutation to explore the relationship between Gatc and other components .

The GATC genetic algorithm offers several advantages:

• Efficient exploration of phylogeny space

• Reduction in the risk of falling into local minima

• Reasonable computational time requirements

• Ability to work with both resolved and unresolved trees

How can researchers address data contradictions in studies involving recombinant Gatc?

When facing contradictory results in studies involving recombinant Gatc, researchers should implement a systematic approach to identify and resolve discrepancies:

Common Sources of Contradiction in Gatc Studies

• Technical variations: Different expression systems (bacterial vs. mammalian) can affect protein folding and activity

• Post-translational modifications: Variations in glycosylation or phosphorylation status between preparations

• Buffer composition effects: Ionic strength, pH, and presence of stabilizing agents

• Experimental design differences: Temperature, incubation time, substrate sources

• Data interpretation frameworks: Statistical approaches and threshold definitions

Structured Resolution Framework

Implement a context validation process similar to that described for RAG (Retrieval-Augmented Generation) systems, which can be adapted for experimental data analysis :

• Contradiction detection: Systematically review experimental data to identify potential inconsistencies

• Categorization of contradiction types:

  • Self-contradictory results within a single experimental setup

  • Contradicting results between different experimental approaches

  • Conditional contradictions where certain conditions create contradictions between otherwise consistent results

• Resolution strategies:

  • Perform additional control experiments to test specific hypotheses about the source of contradiction

  • Implement standardized protocols across research groups

  • Use multiple methodological approaches to triangulate results

Documentation and Reporting

When reporting contradictory findings:

• Clearly state the experimental conditions that led to different outcomes

• Discuss potential mechanistic explanations for the contradictions

• Suggest experimental designs that could resolve the contradictions

• Share raw data to enable independent analysis by other researchers

What are the optimal conditions for expressing and purifying functional recombinant mouse Gatc for structural studies?

Obtaining high-quality recombinant mouse Gatc for structural studies requires careful optimization of expression and purification conditions:

Expression System Selection

Based on available data, E. coli is the preferred expression system for mouse Gatc, as it produces a single, non-glycosylated polypeptide chain suitable for structural studies . Consider the following optimization parameters:

Purification Protocol

For structural studies, multi-step purification is recommended:

• Initial capture: Ni-NTA affinity chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 0.15 M NaCl, 30% glycerol, 1 mM DTT

  • Wash extensively to remove contaminants

  • Elute with imidazole gradient (50-300 mM)

• Secondary purification: Ion exchange chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 50 mM NaCl, 5% glycerol

  • Salt gradient elution (50-500 mM NaCl)

• Polishing step: Size exclusion chromatography

  • Buffer: 20 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 1 mM DTT

  • Analyze fractions by SDS-PAGE for purity assessment

• Concentration: Use molecular weight cut-off filters (10 kDa)

  • Concentrate to 5-10 mg/mL for crystallization trials

  • Add stabilizing agents if needed (0.1% BSA for non-structural applications)

Quality Control for Structural Studies

Before proceeding to structural studies, verify:

• Purity (>95% by SDS-PAGE and mass spectrometry)

• Homogeneity (single peak by dynamic light scattering)

• Proper folding (circular dichroism spectroscopy)

• Biological activity (transamidation activity assay)

How does the function of recombinant mouse Gatc compare to human GATC in research applications?

Understanding the similarities and differences between mouse and human GATC is essential for translational research:

Sequence and Structural Homology

Mouse Gatc shares significant sequence homology with human GATC, making it a suitable model for many research applications. Key comparisons include:

• Mouse Gatc cDNA encodes 223 amino acids including a 25 aa signal peptide and a 198 aa mature protein

• Human GATC contains similar domain organization with high sequence conservation in the catalytic regions

• Both proteins function as part of the heterotrimeric GatCAB complex in mitochondria

Functional Conservation and Differences

Experimental Considerations

When using mouse Gatc as a model for human GATC research:

• For structural studies: Mouse Gatc is an excellent proxy due to high sequence conservation in functional domains

• For interaction studies: Consider species-specific interaction partners that may differ between mouse and human

• For disease modeling: Mouse interference models of Gatc recapitulate key features of human mitochondrial translation disorders

• For drug development: Mouse models provide valuable screening platforms, but validation in human systems is essential

What methodological approaches are recommended for investigating the role of Gatc in mitochondrial disease models?

Investigating the role of Gatc in mitochondrial disease models requires a comprehensive methodological framework:

Disease Model Development

Phenotypic Characterization

For comprehensive phenotypic analysis of Gatc-related mitochondrial disease models:

• Mitochondrial function assessment:

  • Oxygen consumption rate measurements

  • Membrane potential analysis using potentiometric dyes

  • ATP production capacity

  • ROS production quantification

• Molecular analysis:

  • Quantification of mitochondrial translation products

  • Analysis of respiratory chain complex assembly

  • Measurement of mitochondrial DNA copy number

  • Transcriptomics of mitochondrial and nuclear genes

• Cellular consequences:

  • Cell viability and proliferation assessments

  • Apoptosis quantification

  • Mitochondrial morphology and dynamics

  • Metabolomic profiling

Therapeutic Intervention Testing

When testing potential therapeutic interventions in Gatc-deficient models:

• Design rescue experiments with wild-type Gatc expression to confirm phenotype specificity

• Test metabolic bypass strategies that might circumvent the need for fully functional mitochondrial translation

• Evaluate antioxidant treatments to mitigate increased ROS production

• Assess small molecules that might stabilize partially functional GatCAB complexes

• Apply systematic experimental design principles with appropriate controls and sufficient replication

For all therapeutic interventions, establish clear metrics for success based on the reversal of specific molecular, cellular, and physiological phenotypes associated with Gatc dysfunction.