Low temperature solution behaviour of Methylophilus methylotrophus electron transferring flavoprotein: a study by analytical ultracentrifugation
| Autor: | Helmut Cölfen, Emma K. Wilson, Nigel S. Scrutton, Stephen E. Harding, Donald J. Winzor |
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| Rok vydání: | 1997 |
| Předmět: |
biology
Chemistry Biophysics Analytical chemistry General Medicine Electron-transferring flavoprotein Dissociation (chemistry) Analytical Ultracentrifugation Sedimentation coefficient Dissociation constant Crystallography Sedimentation equilibrium Methylophilus methylotrophus biology.protein Molar mass distribution |
| Zdroj: | European Biophysics Journal. 25:411-416 |
| ISSN: | 1432-1017 0175-7571 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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