Conservation of tubulin alpha-beta differences in zinc-induced sheets with variations in pH and...

Zinc-induced tubulin sheets were grown at pH values of 5.7 and 6.4 from porcine brain tubulin purified by phosphocellulose chromatography as well as from microtubule protein containing tubulin plus 20% (w/w) unfractionated microtubule-associated proteins (MAPs). Electron micrographs of negatively stained sheets were analyzed by a combination of real space cross-correlation and Fourier space methods, providing two-dimensional reconstructions to approximately 16 A resolution. The reconstructed images revealed that the protofilaments comprising zinc-induced sheets are composed of two clearly distinguishable alternating subunits, presumably corresponding to the alpha- and beta-tubulin monomers, whose morphologies are not significantly influenced by pH or the presence of MAPs during sheet assembly. Sheets assembled at pH 5.7, either with or without MAPs, were divided into two domains by a protofilament discontinuity which was not present in sheets assembled at pH 6.4, and displayed a 2.4 A reduction of the interprotofilament distance in projection relative to sheets assembled at pH 6.4. We conclude that morphological differences between tubulin subunits represent intrinsic structural features not contributed by MAPs, and that pH is more important than MAP content in influencing the lattice parameters of zinc-induced sheets.