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, Mw,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, Mw,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 Kd. Sedimentation velocity experiments yielded an estimate of the heterodimer sedimentation coefficient, s020,w, of (4.5 ± 0.2) S which for M = 63,700 Da suggests a globular structure.