The diamagnetic susceptibility of the tubulin dimer

Abstract

An approximate value of the diamagnetic anisotropy of the tubulin dimer, Δχ dimer, has been determined assuming axial symmetry and that only the α -helices and β -sheets contribute to the anisotropy. Two approaches have been utilized: (a) using the value for the Δχ α for an α -helical peptide bond given by Pauling (1979) and (b) using the previously determined anisotropy of fibrinogen as a calibration standard. The Δχ dimer ≈ 4 × 10(-27) JT(-2) obtained from these measurements are similar to within 20%. Although Cotton-Mouton measurements alone cannot be used to estimate Δχ directly, the value we measured, CMdimer = (1.41 ± 0.03) × 10(-8) T(-2)cm(2)mg(-1), is consistent with the above estimate for Δχ dimer. The method utilized for the determination of the tubulin dimer diamagnetic susceptibility is applicable to other proteins and macromolecular assemblies as well.

Figure 1

Representative example of Cotton-Mouton measurements on a dimer solution, 4 mg/mL, in Tris buffer. The magnetic field ramp rate was ΔB/ΔT = 50 (T min⁡⁻¹). The results of the up and down sweep of the magnetic field are shown. A linear fit of the data (solid line) was used to obtain the Cotton-Mouton constant. Δn is also expressed in mV to emphasize that these experiments are on the limit of sensitivity of the instrument. The statistical errors can be estimated from the variations of the data with respect to the linear fit.

Figure 2

Cotton-Mouton constants, normalized to a concentration of 1 mg/mL, determined for both dimeric and double dimer samples as function of concentration. The estimated error margin is discussed in the text. The dimer solutions contain only a small, concentration independent fraction of larger aggregates. For the double dimer solutions, the composition is indicated in the figure. “2+” indicates the weight percentage of larger aggregates, “2” the weight percentage of double, dimers and “1” the amount of single dimers.