Recombinant Electron transfer flavoprotein subunit beta (etfB)

What is the structure and function of Electron Transfer Flavoprotein Subunit Beta (ETFB)?

ETFB is one of two subunits that compose the Electron Transfer Flavoprotein (ETF) heterodimer, alongside the alpha subunit (ETFA). The complete ETF protein contains one FAD and one AMP molecule as cofactors. Structurally, ETFB primarily contributes to domain III of the assembled ETF protein, with a small C-terminal portion contributing to domain II. The AMP cofactor is buried deeply within domain III of ETFB, where it forms hydrogen bonds with specific residues including Ala126, Asp129, Asn132, Gln133, and Thr134 .

Functionally, ETFB plays an essential role in the heterodimeric ETF complex that serves as an electron acceptor from at least 14 different flavoenzymes in the mitochondrial matrix. These enzymes are involved in fatty acid β-oxidation and amino acid degradation pathways. ETF subsequently transfers these electrons to ETF:QO (Electron Transfer Flavoprotein-Ubiquinone Oxidoreductase), which feeds them into the respiratory chain at the level of complex III via ubiquinone .

How is ETFB encoded and expressed in humans?

ETFB is nuclear-encoded by the ETFB gene located on chromosome 19q13.3. The gene is transcribed as a 21,220 bp pre-mRNA, which is processed into an 872 bp mature mRNA consisting of 6 exons. The major transcript variant (NM_001985.3) encodes a 27 kDa protein that is synthesized in the cytosol in a form indistinguishable from its mitochondrial form, suggesting it lacks a cleavable mitochondrial targeting sequence .

An alternative transcript variant (NM_001014763.1) has also been identified that uses a downstream transcript start site in intron 1. This variant encodes a protein with a longer and distinct N-terminus compared to the major transcript variant, though its specific biological function remains unknown .

What experimental methods are used to produce recombinant ETFB?

Methodological approach for recombinant ETFB production:

• Gene cloning: The cDNA encoding human ETFB is PCR-amplified and cloned into a suitable expression vector (commonly pET-based vectors for bacterial expression).

• Expression system selection: While E. coli is the most common expression system, mammalian or insect cell systems may be preferred when post-translational modifications are important. For functional studies requiring ETF complex formation, co-expression with ETFA is often necessary.

• Purification strategy: A typical protocol involves:

  • Metal affinity chromatography using His-tagged ETFB

  • Ion exchange chromatography for further purification

  • Size exclusion chromatography to isolate properly folded protein and remove aggregates

• Quality control: Verify recombinant ETFB by:

  • SDS-PAGE to confirm molecular weight (~27 kDa)

  • Western blot with anti-ETFB antibodies

  • Mass spectrometry to confirm protein identity

  • Circular dichroism to assess proper protein folding

  • Fluorescence spectroscopy to confirm FAD incorporation when co-expressed with ETFA

When producing functional ETF heterodimer, co-expression of both subunits is required, as the characteristic greenish fluorescence (emission peak ~490 nm) is a property of the assembled complex rather than individual subunits .

How do post-translational modifications regulate ETFB function?

Post-translational modifications, particularly lysine methylation, play a significant regulatory role in ETFB function. METTL20, a seven-β-strand methyltransferase localized to mitochondria, specifically tri-methylates ETFB at lysines 200 and 203. This methylation has notable effects on ETF activity and mitochondrial metabolism .

Mechanistically, METTL20-mediated methylation decreases the ability of ETF to extract electrons from medium-chain acyl-CoA dehydrogenase (MCAD) and glutaryl-CoA dehydrogenase in vitro. Studies using knockout models have revealed that absence of METTL20 (and consequently, absence of ETFB methylation) results in:

• Higher ETF activity in mitochondria

• Increased β-oxidation capacity

• Higher oxygen consumption and heat production, particularly when β-oxidation is highly activated (e.g., during ketogenic diet feeding)

• Improved ability to maintain body temperature in cold environments after fasting

These findings indicate that METTL20-mediated ETFB methylation serves as a negative regulator of ETF activity, potentially allowing for metabolic flexibility in response to varying energy demands. Researchers studying ETFB should consider the methylation status of their recombinant proteins, as this may significantly impact functional assays .

What are the technical challenges in expressing functional recombinant ETFB?

Producing functional recombinant ETFB presents several technical challenges that researchers should address:

• Heterodimer formation necessity: ETFB alone is not functional; it must form a heterodimer with ETFA. Studies requiring functional ETF typically necessitate co-expression of both subunits.

• Cofactor incorporation: The ETF complex contains one FAD and one AMP molecule. Ensuring proper cofactor incorporation during recombinant expression is essential for obtaining functional protein. While the AMP cofactor does not directly influence enzymatic activity, it is important for proper assembly of the holo structure .

• Post-translational modifications: Native ETFB undergoes methylation at lysines 200 and 203, which regulates its activity. E. coli expression systems lack the machinery for these modifications, potentially resulting in proteins with different activity profiles than native ETF .

• Protein-protein interaction preservation: ETF interacts with at least 14 different flavoenzymes plus ETF:QO. Ensuring that recombinant ETFB maintains proper interaction interfaces is crucial for functional studies.

• Stability considerations: The ETF complex has a specific structural arrangement with three distinct domains. Domain III, primarily comprising ETFB, contains the AMP binding site. Improper folding or destabilization can occur if recombinant production disrupts these domains.

To address these challenges, researchers often employ co-expression systems for both ETF subunits, supplement growth media with riboflavin to enhance FAD availability, and carefully optimize buffer conditions to maintain protein stability and cofactor association.

How can researchers design assays to measure recombinant ETFB activity in the context of ETF complex?

Designing robust assays for recombinant ETFB activity requires consideration of its physiological role as part of the ETF complex. Several methodological approaches are available:

• Fluorometric assay: This leverages the unique fluorescence properties of ETF (emission peak ~490 nm), which is 3.5 times higher than free FAD. The fluorescence is quenched upon reduction, enabling real-time monitoring of electron transfer activity. Recent improvements allow adaptation to a microplate format, facilitating higher throughput analysis .

  Protocol outline:

  • Mix recombinant ETF with acyl-CoA substrates and purified acyl-CoA dehydrogenases

  • Monitor fluorescence decrease at 490 nm as ETF becomes reduced

  • Calculate reaction rates based on fluorescence quenching kinetics

• Spectrophotometric assays: These track ETF reduction by measuring absorbance changes at characteristic wavelengths.

  Protocol outline:

  • Oxidized ETF exhibits maxima at 373 nm and 436 nm

  • Reduction forms an anionic semiquinone with a characteristic spectrum

  • Monitor absorbance changes at these wavelengths during interaction with electron donors

• Oxygen consumption assays: These measure downstream effects on the respiratory chain.

  Protocol outline:

  • Use an oxygen electrode or Seahorse analyzer to measure oxygen consumption rates

  • Add substrates that feed electrons to ETF via specific dehydrogenases

  • Compare rates with and without functional ETF complex

• Reconstituted electron transfer system: This comprehensive approach measures complete electron flow.

  Protocol outline:

  • Combine recombinant ETF, purified dehydrogenases, recombinant ETF:QO, and artificial electron acceptors (e.g., Q1 or ferricenium)

  • Initiate reaction with appropriate substrates (e.g., acyl-CoAs)

  • Monitor reduction of terminal electron acceptors spectrophotometrically

When interpreting results, researchers should consider the methylation status of ETFB, as METTL20-mediated methylation at lysines 200 and 203 significantly decreases electron transfer rates from certain dehydrogenases .

What are the genetic variants of ETFB and their implications for research?

Understanding ETFB genetic variants is crucial for comprehensive research, as these variants can affect protein function, stability, and pathogenicity. The main categories include:

Researchers may need to express and characterize multiple ETFB variants to fully understand their functional implications in different experimental contexts. When selecting a variant for expression, consider the specific research question and whether native interaction partners will be from the same species to ensure biologically relevant results.

How can researchers optimize the purification of recombinant ETFB to ensure proper complex formation with ETFA?

Optimizing purification of recombinant ETFB for proper complex formation with ETFA requires a strategic approach focusing on co-expression, cofactor incorporation, and complex stability:

• Co-expression strategies:

  • Dual vector system: Express ETFA and ETFB from separate vectors with different antibiotic selection markers

  • Bicistronic vector: Express both subunits from a single vector with an internal ribosome entry site (IRES)

  • Fusion protein approach: Express as a fusion protein with a cleavable linker, followed by in vitro processing

• Optimized purification protocol:

  • Cell lysis: Use gentle methods (e.g., freeze-thaw cycles or mild detergents) to preserve native-like subunit interactions

  • Affinity tags placement: Consider differential tagging (e.g., His-tag on one subunit, GST-tag on the other) to ensure purification of complete complexes only

  • Sequential purification: First capture the complex using one affinity tag, then verify complex integrity using the second tag

  • Native elution conditions: Use mild elution conditions to maintain complex integrity

  • Size exclusion chromatography: Essential final step to isolate properly assembled heterodimers from individual subunits or aggregates

• Quality control measures:

  • Spectroscopic verification: The ETF complex has characteristic absorption peaks at 373 nm and 436 nm when oxidized

  • Fluorescence analysis: Native-like ETF complex exhibits ~3.5 times higher fluorescence than free FAD (emission peak ~490 nm)

  • Activity assays: Verify electron transfer capability from model dehydrogenases

  • Analytical ultracentrifugation: Confirm proper heterodimer formation with expected molecular weight (~58 kDa)

• Cofactor supplementation:

  • Add riboflavin to expression media to ensure adequate FAD availability

  • Include AMP in purification buffers to maintain proper cofactor association, as AMP is important for stability of the holo structure

Successful purification results in a stable heterodimeric complex with characteristic spectroscopic properties and electron transfer capabilities.

What are the best experimental models to study ETFB function in the context of metabolic disorders?

Research into ETFB function in metabolic disorders benefits from diverse experimental models, each offering specific advantages for understanding different aspects of ETFB biology:

• Cell-free systems:

  • Reconstituted electron transfer assays: Combining purified recombinant ETFB, ETFA, ETF:QO, and various dehydrogenases to study electron transfer kinetics and the effects of specific mutations

  • Advantages: Precise control over components, quantitative kinetic measurements

  • Applications: Structure-function studies, drug screening, mechanistic investigations

• Cellular models:

  • Patient-derived fibroblasts: Primary cells from MADD patients carrying ETFB mutations

  • CRISPR-modified cell lines: Engineered to carry specific ETFB variants or knockouts

  • Advantages: Preserved cellular context, ability to study metabolic consequences

  • Applications: Cellular phenotyping, rescue experiments with recombinant ETFB, drug screening

• Animal models:

  • Knockout mice: Complete Mettl20 knockout mice have revealed the regulatory role of ETFB methylation in vivo

  • Key findings: METTL20 knockout mice showed higher ETF activity, increased β-oxidation capacity, higher oxygen consumption, and better temperature maintenance in cold environments after fasting

  • Advantages: Complex physiological responses can be studied

  • Applications: Whole-body metabolism studies, therapeutic testing

• Tissue-specific approaches:

  • Ex vivo tissue studies: Particularly using liver, muscle, or adipose tissue samples

  • Tissue-specific knockouts: Creating tissue-specific ETFB knockouts or mutant expression

  • Advantages: Reveals tissue-specific functions and compensatory mechanisms

  • Applications: Understanding tissue-specific manifestations of ETFB defects

• Metabolomic profiling:

  • Targeted metabolomics: Measuring specific acylcarnitines, organic acids, and other metabolites affected by ETF dysfunction

  • Advantages: Provides functional readout of metabolic consequences

  • Applications: Biomarker discovery, monitoring treatment efficacy

Researchers typically employ multiple models in complementary fashion, with recombinant ETFB often used in rescue experiments or for structural studies to understand the molecular basis of observed phenotypes.

How can researchers measure the interaction between recombinant ETFB and its various dehydrogenase partners?

Measuring interactions between recombinant ETFB (as part of the ETF complex) and its various dehydrogenase partners requires techniques that can detect, quantify, and characterize protein-protein interactions with varying affinities. Recommended methodological approaches include:

• Biophysical interaction analysis:

  • Surface Plasmon Resonance (SPR): Quantifies binding kinetics and affinities in real-time

    • Immobilize either ETF or dehydrogenase partner on sensor chip

    • Flow partner protein at varying concentrations

    • Determine kon, koff, and KD values

  • Isothermal Titration Calorimetry (ITC): Measures thermodynamic parameters of binding

    • Provides enthalpy (ΔH), entropy (ΔS), and binding stoichiometry

    • Particularly useful for comparing wild-type and mutant interaction profiles

  • Microscale Thermophoresis (MST): Detects binding through changes in thermophoretic mobility

    • Requires minimal protein amounts

    • Works well for transient interactions

• Structural approaches:

  • Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS): Maps interaction interfaces

    • Identifies protected regions upon complex formation

    • Provides insights into conformational changes

  • Cryo-Electron Microscopy: Visualizes complexes at near-atomic resolution

    • Has been used successfully for similar electron transfer complexes

    • Can capture different conformational states

• Functional interaction assays:

  • Electron transfer kinetics: Direct measurement of functional interaction

    • Monitor rate of dehydrogenase-dependent ETF reduction

    • Compare kinetic parameters (kcat, Km) across different partners

  • Protein crosslinking: Captures transient interactions

    • Use chemical crosslinkers followed by mass spectrometry

    • Identifies specific residues involved in the interaction

• Computational approaches:

  • Molecular docking: Predicts interaction modes

  • Molecular dynamics simulations: Models dynamic aspects of interaction

A comprehensive experimental approach incorporating multiple techniques provides the most reliable characterization of ETFB interactions. Importantly, researchers should consider the regulatory role of post-translational modifications such as the tri-methylation of ETFB at lysines 200 and 203 by METTL20, which has been shown to decrease the ability of ETF to extract electrons from certain dehydrogenases .

What analytical techniques are most effective for characterizing post-translational modifications of ETFB?

Characterizing post-translational modifications (PTMs) of ETFB, particularly the regulatory methylation at lysines 200 and 203, requires sophisticated analytical approaches:

• Mass Spectrometry-Based Techniques:

  • Bottom-up proteomics:

    • Enzymatic digestion of ETFB followed by LC-MS/MS analysis

    • Peptide mass shifts indicate modifications (e.g., +14 Da per methyl group)

    • Fragmentation patterns confirm modification sites

  • Top-down proteomics:

    • Analysis of intact ETFB protein

    • Provides comprehensive view of combinatorial modifications

    • Reveals relative abundance of different PTM states

  • Selected/Multiple Reaction Monitoring (SRM/MRM):

    • Targeted approach for quantifying specific modified peptides

    • Enables precise quantification of methylation stoichiometry

    • Useful for comparing methylation levels under different conditions

• Immunological Methods:

  • Western blotting with modification-specific antibodies:

    • Antibodies against tri-methylated K200/K203 of ETFB

    • Provides relative quantification across samples

    • Useful for screening multiple samples rapidly

  • Immunoprecipitation followed by MS:

    • Enriches for modified forms of ETFB

    • Increases detection sensitivity for low-abundance PTMs

• Functional Correlation Analysis:

  • Site-directed mutagenesis followed by activity assays:

    • Replace K200/K203 with residues that cannot be methylated (e.g., arginine)

    • Compare electron transfer activity with wild-type ETFB

    • Determines functional significance of specific modifications

  • In vitro methylation assays:

    • Recombinant METTL20 can be used to methylate ETFB in vitro

    • Allows controlled studies of methylation effects on activity

• Structural Analysis of Modified ETFB:

  • X-ray crystallography or Cryo-EM:

    • Reveals structural impacts of methylation

    • Can identify potential allosteric effects

  • Hydrogen-Deuterium Exchange MS:

    • Detects conformational changes induced by methylation

    • Maps regions with altered solvent accessibility

When applying these techniques, researchers should consider that ETFB methylation by METTL20 has been shown to decrease ETF's ability to extract electrons from medium-chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase. Studies with METTL20 knockout mice have demonstrated that absence of this methylation results in higher ETF activity, increased β-oxidation capacity, and better temperature maintenance in cold environments after fasting .

How can recombinant ETFB be used to investigate Multiple Acyl-CoA Dehydrogenase Deficiency (MADD)?

Recombinant ETFB serves as a powerful tool for investigating Multiple Acyl-CoA Dehydrogenase Deficiency (MADD), a rare metabolic disorder caused by mutations in ETFA, ETFB, or ETFDH genes. Strategic applications include:

• Functional characterization of patient mutations:

  • Expression and purification of mutant ETFB proteins:

    • Generate recombinant ETFB proteins with patient-specific mutations

    • Co-express with wild-type ETFA to form heterodimeric complexes

    • Evaluate protein stability, folding, and cofactor binding

  • Biochemical activity assessment:

    • Measure electron transfer rates from various dehydrogenases

    • Quantify interaction affinities with partner proteins

    • Determine enzyme kinetic parameters (kcat, Km)

  • Results interpretation:

    • Classify mutations as affecting protein stability, cofactor binding, or catalytic activity

    • Correlate biochemical defects with clinical severity

    • Identify potential genotype-phenotype correlations

• Development of therapeutic approaches:

  • Protein replacement screening:

    • Test modified recombinant ETFB variants for improved stability or activity

    • Evaluate cell-penetrating peptide fusions for protein delivery

    • Assess activity restoration in patient-derived cells

  • Small molecule screening:

    • Identify compounds that can stabilize mutant ETFB

    • Develop high-throughput assays using recombinant proteins

    • Validate hits in cellular and animal models

• Diagnostic applications:

  • Development of functional assays:

    • Design biochemical tests to measure ETF activity in patient samples

    • Create reference standards using recombinant proteins

    • Establish correlations between assay results and disease severity

• Structure-guided insights:

  • Mapping mutation locations on ETF structure:

    • Visualize how mutations affect critical regions of the protein

    • Identify potential hotspots for therapeutic intervention

    • Guide rational design of stabilizing modifications

By systematically applying recombinant ETFB in these research areas, investigators can gain mechanistic insights into MADD pathophysiology and develop targeted therapeutic strategies based on the specific molecular defects associated with different mutations .

What role does ETFB play in mitochondrial metabolism regulation during different metabolic states?

ETFB plays a pivotal regulatory role in mitochondrial metabolism across various metabolic states, primarily through its function in the ETF complex and its post-translational modification status:

This regulatory role positions ETFB as a potential therapeutic target for metabolic disorders characterized by dysregulated fatty acid oxidation or mitochondrial function.

What emerging technologies could advance our understanding of ETFB regulation and function?

Several cutting-edge technologies show promise for advancing our understanding of ETFB regulation and function:

These emerging technologies could particularly enhance our understanding of how METTL20-mediated methylation of ETFB at lysines 200 and 203 regulates mitochondrial metabolism under different physiological conditions, potentially revealing new therapeutic approaches for metabolic disorders .

What are the implications of ETFB methylation for developing therapeutics targeting mitochondrial metabolism?

The discovery that METTL20-mediated methylation of ETFB regulates ETF activity and mitochondrial metabolism opens several promising avenues for therapeutic development:

• Targeting METTL20 activity for metabolic disorders:

  • Inhibiting METTL20: Could potentially increase β-oxidation capacity and energy expenditure

    • Potential applications: Obesity, metabolic syndrome, hepatic steatosis

    • Mechanism: Reduced ETFB methylation would enhance ETF activity, promoting fat utilization

  • Enhancing METTL20: Might help regulate excessive β-oxidation

    • Potential applications: Certain inborn errors of metabolism with toxic buildup of fatty acid intermediates

    • Mechanism: Increased ETFB methylation would moderate ETF activity

• Therapeutic potential in cold stress and thermogenesis:

  • METTL20 knockout mice demonstrated better ability to maintain body temperature in cold environments after fasting

  • This suggests that ETFB methylation status influences thermal regulation

  • Potential applications:

    • Hypothermia prevention in vulnerable populations

    • Enhancement of cold-induced thermogenesis for metabolic health

    • Management of torpor-like states in critical care settings

• Precision medicine approaches for MADD:

  • Different ETFB mutations might respond differently to methylation status

  • Personalized therapeutic strategies could be developed based on:

    • Specific mutation location and type

    • Effect of methylation on mutant protein stability or activity

    • Patient-specific metabolic profile

• Development of biomarkers based on ETFB methylation status:

  • ETFB methylation levels could serve as biomarkers for:

    • Mitochondrial function in various disease states

    • Predicting response to metabolic interventions

    • Monitoring treatment efficacy

• Combinatorial approaches with existing therapies:

  • METTL20 modulation could potentially enhance effects of:

    • Currently used metabolic modulators (e.g., fibrates, thiazolidinediones)

    • Dietary interventions like ketogenic diets

    • Exercise-based therapies for metabolic disorders

The discovery that METTL20 knockout mice show higher oxygen consumption and heat production, especially when fed a ketogenic diet, suggests that ETFB methylation status could be particularly relevant for therapeutic approaches targeting fat utilization and energy expenditure in metabolic disorders .

What are common pitfalls in working with recombinant ETFB and how can researchers overcome them?

Researchers working with recombinant ETFB face several common challenges that require specific troubleshooting strategies:

• Expression and solubility issues:

  • Challenge: ETFB may form inclusion bodies when expressed alone in bacterial systems

  • Solution:

    • Co-express with ETFA to promote proper folding and complex formation

    • Use lower induction temperatures (16-18°C) and reduced IPTG concentrations

    • Consider fusion tags that enhance solubility (e.g., SUMO, MBP)

    • Try insect cell or mammalian expression systems for problematic constructs

• Heterodimer stability problems:

  • Challenge: The ETF heterodimer may dissociate during purification

  • Solution:

    • Include stabilizing agents in buffers (glycerol 10-20%, low concentrations of detergents)

    • Consider chemical crosslinking for structural studies

    • Use tandem affinity purification with tags on both subunits

    • Monitor complex integrity by size exclusion chromatography throughout purification

• Cofactor incorporation difficulties:

  • Challenge: Incomplete FAD incorporation leading to heterogeneous preparations

  • Solution:

    • Supplement expression media with riboflavin

    • Add FAD during cell lysis and purification steps

    • Include a reconstitution step with excess FAD followed by removal of unbound cofactor

    • Verify FAD content spectrophotometrically (A373/A450 ratio)

• Activity measurement inconsistencies:

  • Challenge: Variable activity results due to different methylation states or partner protein effects

  • Solution:

    • Carefully characterize methylation status of recombinant preparations using mass spectrometry

    • Consider co-expression with or without METTL20 to control methylation

    • Design experiments to account for ETFB methylation status when interpreting activity data

    • Use consistent sources of partner proteins (dehydrogenases) for comparable results

• Interference from post-translational modifications:

  • Challenge: Bacterial expression systems lack the machinery for proper ETFB methylation

  • Solution:

    • For studies requiring native-like methylation, use mammalian expression systems

    • Perform in vitro methylation using recombinant METTL20

    • Create methylation-mimicking mutants (e.g., K→R to prevent methylation or K→Q to mimic aspects of methylation)

• Storage stability issues:

  • Challenge: Activity loss during storage

  • Solution:

    • Store at high concentration (>1 mg/ml) with stabilizers (glycerol, reducing agents)

    • Flash-freeze aliquots in liquid nitrogen

    • Avoid repeated freeze-thaw cycles

    • Verify activity before experiments using standardized assays

Understanding that ETFB functions as part of a complex with specific post-translational modifications, particularly methylation at lysines 200 and 203, is crucial for designing experiments and interpreting results correctly .

How can researchers integrate structural and functional studies of ETFB to gain comprehensive insights?

Integrating structural and functional studies of ETFB requires a multidisciplinary approach that connects molecular structure to biological function:

• Structure-guided functional analysis:

  • Static structural studies:

    • X-ray crystallography of ETF complexes with different partners

    • Cryo-EM to capture various conformational states

    • NMR to identify dynamic regions and binding interfaces

  • Structure-informed mutations:

    • Design mutations at key interfaces identified from structures

    • Create ETFB variants with altered:

      • Domain interactions

      • FAD binding properties

      • Methylation sites (K200/K203)

      • Partner protein interaction surfaces

  • Functional validation:

    • Measure electron transfer kinetics of mutant proteins

    • Quantify binding affinities with partner proteins

    • Assess effects on mitochondrial respiration in cellular models

• Dynamic structural approaches:

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

    • Map conformational changes upon:

      • Partner protein binding

      • Redox state changes

      • METTL20-mediated methylation

  • Molecular dynamics simulations:

    • Model conformational dynamics of wild-type vs. mutant ETFB

    • Simulate effects of methylation on protein flexibility and interaction surfaces

  • Correlate with function:

    • Connect identified dynamic regions with electron transfer efficiency

    • Link conformational changes to regulatory mechanisms

• Integrated cellular studies:

  • CRISPR-engineered cellular models:

    • Generate cells with structure-informed ETFB mutations

    • Create methylation-deficient variants (K200R/K203R)

  • Functional readouts:

    • Measure fatty acid oxidation rates

    • Quantify oxygen consumption and ATP production

    • Assess cellular response to metabolic challenges

  • Structure-function correlation:

    • Connect molecular perturbations to cellular phenotypes

    • Validate structural hypotheses in cellular context

• High-throughput screening informed by structure:

  • Design focused compound libraries:

    • Target specific structural features or interfaces

    • Aim to modulate ETFB methylation or partner interactions

  • Activity-based screening:

    • Develop assays based on structural understanding

    • Screen for compounds that affect specific ETFB functions

  • Structural validation of hits:

    • Confirm binding modes of active compounds

    • Iterate structure-based optimization

This integrated approach has already yielded valuable insights, such as understanding how METTL20-mediated methylation of ETFB at lysines 200 and 203 reduces ETF's ability to extract electrons from certain dehydrogenases, thereby regulating mitochondrial metabolism and heat production—an effect that becomes particularly important during metabolic stress conditions such as cold exposure after fasting .