Schlieper et al. 10.1073/pnas.0502859102. |
Fig. 5.
BtubA/B is monomeric at low concentrations and associates into dimers at higher concentrations as indicated by sedimentation equilibrium analysis. (A) The sedimentation equilibrium profile of BtubA/B (7.2 m M; solid circles and left ordinate scale) gave an average molecular weight of 53,500 ± 360 (solid line fit), slightly above the 48,841 theoretical value of the average A/B monomer molecular weight. However, BtubA/B at a higher concentration (49.5 m M; void circles and right ordinate scale) gave a more steep concentration gradient corresponding to an apparent molecular weight of 72,260 ± 480 (dashed line fit), with upwardly curved residuals (see upper plot), which is indicative of an association process. Conditions were 100 mM Pipes•NaOH/200 mM KCl (pH 6.8) at 20°C and 10,000 rpm. (B) The dependence of the average molecular weight of BtubA/B on protein concentration confirmed a weak association of A/B monomers to dimers. The data obtained from BtubA/B solutions of concentrations ranging between 7.2 and 88 m M (conditions as above) were used to obtain local molecular weight values, which are plotted vs. protein concentration. The solid line is a best model fit to an association scheme nA Û An, where the molecular weight of the monomer is 47,500, the association degree is n = 2.5, and the equilibrium association constant is K » 104 M–1. A similar experiment performed in 20 mM Tris•HCl (pH 7.1) was best model fitted by monomer weight 44,500, n = 1.9, and K » 105 M–1.
Supporting Materials and Methods
Sedimentation equilibrium measurements were made with a Beckman Optima XL-A ultracentrifuge with absorption optics, by using short liquid columns in six-channel centerpieces at 10,000, 12,000, and 14,000 rpm at 20°C, and the baselines were measured after centrifugation at 43,000 rpm. The partial specific volume of BtubA/B was 0.736 ml•g–1 (calculated from the amino acid sequence). The sedimentation equilibrium data were analyzed as reported in ref. 1. The association of BtubA/B was model analyzed, in a first approximation, as a homo-association of an average A/B monomer with extinction coefficient 43,275 M–1•cm–1 (280 nm).
1. Rivas, G., Lopez, A., Mingorance, J., Ferrandiz, M. J., Zorrilla, S., Minton, A. P., Vicente, M. & Andreu, J. M. (2000) J. Biol. Chem. 275, 11740–11749.