Microtubules are cylindrical organelles which play critical roles in mitosis and other cell processes. Because of their role in mitosis, microtubules are the target of taxol, vinblastine and other anti-tumor drugs. Microtubules are composed of the protein tubulin, consisting of two subunits called a and b. Both of these exist in numerous isotypic forms. The different forms of b have been highly conserved in evolution and appear to be functionally different. One of these, bIII , is normally found in neurons and steroili cells, but occurs in elevated amounts in a variety of tumors as well. It also has significantly different interactions with the anti-tumor drugs vinblastine, taxol and colchicine. Part of the reason for the uniqueness of the abIII dimer is the apparent unusual rigidity of its conformation. abIII may be an excellent target for rational drug design, for which it is necessary to determine its three-dimensional structure. However, tubulin is a highly unusual protein in that it has very flexible conformation. The overall aim of this grant is to identify the conformational changes that tubulin undergoes, both spontaneously and in the presence of anti-tumor drugs, to compare these changes in different isotypes and to correlate this information with the effects of drugs on microtubule assembly and dynamics. The first specific aim is to analyze the conformational properties of up to four different isotypes (abI, abIII, abIII, and abIV) using a variety of techniques including probes for sulfhydryl groups and hydrophobic areas. The second specific aim will examine the effects of ligands on these conformational properties; these ligands which have been carefully chosen in that each one has a unique set of interactions with tubulin are colchicine, podophyllotoxin, curacin A, vinblastine, maytansine, phompsin, IK104, griseofulvin, BisANs, taxol, taxotere, GTP, and GDP. In the third specific aim, the specific regions on tubulin affected by these ligands will be identified and located in the known sequence of tubulin. In collaborative studies (fourth specific aim), attempts will be made to furnish the three dimensional structure of tubulin. The results of this study should provide a much clearer understanding of the specific effects of drugs on the tubulin molecule and microtubule dynamics as well as the interactions of tubulin isotypes with anti-tumor drugs.
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