Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: A study by analytical ultracentrifugation

The solution behaviour of electron transferring flavoprotein (ETF) from Methylophilus methylotrophus was investigated at low temperature (4°C) by analytical ultracentrifugation. The concentration dependence of the apparent weight average molecular weight, M(w,app), established the existence of the protein in heterodimeric state (M=63,700 Da), but also signified the possible dissociation of the heterodimer at lower concentrations into its constituent subunits (M = 28,900 Da and 33,700 Da, together with FAD and AMP cofactors of collective M = 1120 Da). This similarity in subunit size allows approximate quantification of the dissociation in terms of expressions for a monomer- dimer equilibrium. The dissociative behaviour was confirmed by determination of the point average molecular weight, M(w,app)(r), as a function of the ETF concentration, c(r), throughout the sedimentation equilibrium distributions obtained with loading concentrations of 0.4 and 0.7 mg/ml. By means of the recently formulated 'psi' procedure for direct analysis of solute self- association a value of (1.5±0.1) μM has been obtained for the dissociation constant K(d). Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s(20,w)/⁰ of (4.5±0.2) S which for M=63,700 Da suggests a globular structure.