MOLECULAR MOBILITY OF NITROXYL-LABELED TAXOL DURING TUBULIN ASSEMBLY

Taxol(1) is an important natural anticancer agent that binds to beta-t ubulin and suppresses microtubule depolymerization, We have used elect ron paramagnetic resonance (EPR) spectroscopy to analyze the molecular motion of three novel nitroxyl free radical taxol analogues, Taxol wa s chemically modified at C2 or C7 carbon of the taxane ring with the T EMPO free radical to yield two spin-labelled taxols and concurrently a t C2' and 3'N of the side chain to yield a spin-labelled taxol biradic al, Nitroxyl moieties attached to the taxane ring are significantly re stricted in their molecular motion during microtubule assembly, and th ey 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 diner structure, However, the taxane ring is strong ly immobilized after polymerization of tubulin, i.e. it is incorporate d 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 assem bly of microtubules, and no differences associated with the process of polymerization were detected, The taxol side chain resides in a regio n that is not significantly constrained during polymerization. (C) 199 7 Federation of European Biochemical Societies.