Stabilization of tubulin by deuterium oxide

Tubulin is an unstable protein when stored in solution and loses its abilit y to form microtubules rapidly. We have found that D2O stabilizes the prote in against inactivation at both 4 and 37 degrees C. In H2O-based buffer, tu bulin was completely inactivated after 40 h at 4 degrees C, but in buffer p repared in D2O, no activity was lost after 54 h. Tubulin was completely ina ctivated at 37 degrees C in 8 h in H2O buffer, but only 20% of the activity was lost in D2O buffer. Tubulin also lost its colchicine binding activity at a slower rate in D2O. The deuterated solvent retarded an aggregation pro cess that occurs during incubation at both temperatures. Inactivation in H2 O buffer was partially reversed by transferring the protein to D2O buffer; however, aggregation was not reversed. The level of binding of BisANS, a pr obe of exposed hydrophobic sites in proteins, increases during the inactiva tion of tubulin. In D2O, the rate of this increase is slowed somewhat. We p ropose that D2O has its stabilizing effect on a conformational step or step s that involve the disruption of hydrophobic forces. The conformational cha nge is followed by an aggregation process that cannot be reversed by D2O. A s reported previously Cite, T., and Sate, H. (1984) Biochim. Biophys. Acta 800, 21-27], we found that D2O stimulates the formation of microtubules fro m tubulin. We also observed that the products of assembly in D2O/8% DMSO co nsisted of a high percentage of ribbon structures and incompletely folded m icrotubules. When these polymers were disassembled and reassembled in H2O/8 % DMSO, the products were microtubules. We suggest that the combination of D2O and DMSO, both stimulators of tubulin assembly, leads to the rapid prod uction of nuclei that lead to the formation of ribbon structures rather tha n microtubules.