Microelectrospray ionization analysis of noncovalent interactions within the electron transferring flavoprotein

Area of Science:

• Biochemistry
• Mass Spectrometry
• Structural Biology

Background:

• Electron transferring flavoprotein (ETF) is crucial for cellular metabolism.
• Understanding cofactor binding is essential for elucidating ETF function.
• Previous studies relied on static structural data.

Purpose of the Study:

• To investigate real-time cofactor associations within ETF using muESI-MS.
• To compare cofactor dissociation behavior between porcine (pETF) and human (hETF) ETF.
• To reconcile mass spectrometry findings with existing crystal structure data.

Main Methods:

• Microelectrospray ionization-mass spectrometry (muESI-MS) was employed for real-time analysis.
• Analysis was performed on both porcine and human ETF.
• Varying muESI-MS source energies and solvent conditions (methanol) were utilized.

Main Results:

• Holoprotein was initially observed, but increased energy led to AMP dissociation from both pETF and hETF.
• Analysis in methanol revealed intact subunits and beta-subunits bound to AMP, FAD, or both.
• pETF showed a strong association between the beta-subunit and FAD, with AMP dissociating more readily than FAD.

Conclusions:

• muESI-MS provides dynamic insights into noncovalent cofactor interactions within ETF.
• ETF cofactor binding differs from predictions based on crystal structures.
• muESI-MS complements other biophysical techniques for studying protein-ligand complexes.