Interactions of tubulin and microtubule-associated proteins. Conformation and stability of the oligomeric species from glycerol-cycled microtubule protein of bovine brain

Abstract

The conformation of bovine microtubule protein prepared by cycles of assembly and disassembly in the presence of glycerol was studied by near-UV circular dichroism (c.d.) over a range of protein concentrations. The effects on the conformational properties of ionic strength and of a pH range from 6 to 7.5 were correlated with the known oligomeric composition of microtubule protein prepartions, as determined by the sedimentation behavior of this preparation. The formation of 30 S oligomeric ring species, either by decreasing ionic strength at pH 6.5 or by changing pH in the presence of 0.1 M-NaCl, correlates with a significant change in tubulin c.d. Formation of 18 S oligomer by changing pH at ionic strength 0.2 produced no comparable effect. The c.d. of tubulin dimer itself is not affected by ionic strength and pH over the same range. The results are interpreted as a small conformational adjustment between tubulin and specific microtubule-associated proteins on forming 30 S oligomeric species, due to interaction with the high-molecular-weight-group proteins. The possible significance of this is discussed with respect to microtubule assembly in vitro. By using this conformational parameter, together with equilibrium and kinetic light-scattering studies, the sensitivity of glycerol-cycled microtubule protein to dilution is shown to be strongly pH-dependent, the oligomers being much more stable at pH 6.4 than at pH 6.9. Oligomeric complexes of tubulin with microtuble-associated proteins show marked stability under conditions similar to those for efficient microtubule assembly in vitro. Oligomeric material therefore must be incorporated directly during assembly in vitro from microtubule protein.