Studies of crystallization conditions for native and subtilisin-cleaved pig brain tubulin

A survey of crystallization conditions for pig brain tubulin, using standard vapor diffusion techniques in sitting drops or capillaries, has resulted in irregular, fragile needles or plates with a largest dimension of 0.5 mm. These occurred in 2.5% PEG (MW 3350), 0.1 M Pipes, pH 6.2 and 6.4, 2-16 mM MgSO4, 10-15 mM DTE, and 0.1 mM GDP at 8 degrees C. When GTP replaced GDP these aggregates did not form under any of the conditions surveyed (temperature: 8-10 degrees C; MgSO4: 2-16 mM; pH 6-7; PEG, MW 3350: 1.25-12.5%). EM observations demonstrated that sheets of rings appear in crystal solutions in the presence of GDP or GTP. These results are consistent with the results of Howard and Timasheff (1986, Biochemistry 25, 8292-8300) that tubulin rings form in the presence of GDP or GTP but more readily in GDP. Tubulin crystallization experiments are hampered by tubulin's high degree of heterogeneity. Much of the variability lies in the carboxyl terminal region. Conditions for limited digestion of the heterodimer by subtilisin, removing only the carboxyl terminus, were determined. Reduction of heterogeneity was demonstrated by isoelectric focusing. The solubilities of native and subtilisin-cleaved tubulin in MgSO4, (NH4)2SO4, PEG (MW 1450, 3350, 10,000), DMSO, and MPD were compared. Subtilisin-cleaved tubulin precipitated more readily than native tubulin under all conditions surveyed, consistent with the removal of the highly acidic carboxyl terminus. Vapor diffusion experiments using subtilisin-cleaved tubulin under conditions where native tubulin forms needles or plates resulted in similar aggregates.