Molecular mobility of nitroxyl-labelled taxol during tubulin assembly

Taxol is an important natural anticancer agent that binds to beta-tubulin and suppresses microtubule depolymerization. We have used electron paramagnetic resonance (EPR) spectroscopy to analyze the molecular motion of three novel nitroxyl free radical taxol analogues. Taxol was chemically modified at C2 or C7 carbon of the taxane ring with the TEMPO free radical to yield two spin-labelled taxols and concurrently at C2' and 3'N of the side chain to yield a spin-labelled taxol biradical. Nitroxyl moieties attached to the taxane ring are significantly restricted in their molecular motion during microtubule assembly, and they show no molecular restriction upon binding to tubulin. We conclude that taxol binds to tubulin in a way such that the taxane ring is not constrained by the dimer structure. However, the taxane ring is strongly immobilized after polymerization of tubulin, i.e. it is incorporated into the structure of microtubule. In contrast, the nitroxy moieties of the taxol biradical show strong immobilization upon attachment to tubulin. The nitroxyl energy exchange is restricted prior to the assembly of microtubules, and no differences associated with the process of polymerization were detected. The taxol side chain resides in a region that is not significantly constrained during polymerization.