9406242 Hastie Microtubules are an integral part of the cytoskeleton of all eukaryotic cells. Composed primarily of the protein tubulin, a 100 Kdal heterodimer, these structures perform diverse functions in vivo. Many aspects of the mechanism by which microtubules assemble and disassembly in vitro are currently being unraveled; however, an understanding of microtubule dynamics in vivo remains Microtubule assembly can be perturbed by a variety of exogenous substances. The oldest and most studied compound in this class is colchicine, an alkaloid from the Autumn crocus. Colchicine binding to the soluble tubulin heterodimer results in disruption of normal microtubule assembly and subsequently microtubule-mediated processes. The ubiquity and specificity of colchicine binding to tubulins in higher eukaryotes has led to the hypothesis that the colchicine binding site is of critical importance in the in vivo regulation of microtubule dynamics. To understand the role of the colchicine binding site on tubulin in the processes of microtubule assembly and disassembly, structural information about tubulin and the colchicine binding site is necessary. In this project, the two dimensional structure of the colchicine binding site will be identified through photoaffinity labeling. The relationship of the colchicine binding site and other important domains of the protein in three dimensions will be determined using fluorescence techniques. And finally, the depth of the binding pocket and the nature of the interactions between tubulin and ligands bound to the colchicine site will be probed using l9F NMR spectroscopy. The combined results of these three projects will provide a clearer understanding of the molecular features of the colchicine site on tubulin and its relationship to the structure of the protein. The linear sequence(s) of tubulin that comprise the colchicine binding site will be identified through photoaffinity labeling. Multiple photoaffinity labeling analogs of colchicine in wh ich the photolabile groups are predicted to be within the interior of the colchicine binding site are employed. This approach is innovative in that it is designed to identify peptides within discrete subdomains of the ligand binding site. Moreover, the photolabile groups are an integrated part of the ligand rather than an appendage on the ligand, increasing the likelihood that meaningful results will be obtained. The relationship of the colchicine binding site and the exchangeable GTP site, the Vinca alkaloid binding domain, and the well characterized sulfllydryl moieties on ~-tubulin will be probed in three dimensions by fluorescence resonance energy transfer. These sites are known to be interrelated, but their proximities are unknown. The distances between these sites will be determined using carefully chosen fluorescent probes for the various sites. The results from these investigations will be integrated into the current, low resolution understanding of tubulin structure. The association of ligands with the colchicine site on tubulin appears to be mediated by an unusual, little explored mechanism: a ~-stacking interaction between the ligand and an aromatic amino acid in the binding site, which is probably a tryptophan. The existence of this proposed stacking interaction will be scrutinized by l9F NMR spectroscopy. Like the proton nucleus, the fluorine nucleus is subject to ring current effects that can be observed in the F NMR spectrum. Fluorinated analogs of colchicine will be prepared and their spectra in the presence of tubulin will be determined. These experiments will provide direct evidence of the hypothetical ~-stacking interaction if it exists and will define the region of the binding site in which the stacking interaction takes place. Unlike the proton nucleus, the fluorine nucleus is subject to solvent isotope effects. The fluorinated colchicinoids will be further exploited to determine the solvent accessible and solvent inaccessible portions of the ligands when bound to tubulin. These experiments will serve to delineate the depth of the colchicine binding site on tubulin. tubulin. %%% Microtubules are an integral part of the cytoskeleton of all nucleus-containing cells. These structures are often seen projecting from the nuclear center of the cell to the peripheral wall of the cell and play a major role in cell growth. When a cell divides, the microtubules serve to pull the cell apart to form two daughter cells. Microtubules perform their function in cell division and other types of movement through processes of assembly and disassembly. The microtubules are composed primarily of the protein tubulin, which assembles to form small (13 nm diameter) tubes during cellular division. The assembly and disassembly processes of the purified protein has been studied extensively, but it is unknown how microtubules form and disintegrate in the living cell and what types of factors control their dynamics in the cell. One of the most useful tools for studying microtubules is the alkaloid colchicine. Colchicine is a substance that originates in the Autumn crocus (a member of the lily family). When colchicine binds to tubulin, the colchicine-tublin complex prevents normal assembly of microtubules and thus halts, microtubule-mediated processes. Because colchicine binding activity is found in all animal tubulin, it is thought that the colchicine binding site on tubuli is an important part of the protein in the regulation of microtubule assembly and disassembly in the cell. At this time, little is known about the structure of tubulin and the colchicine binding site on tubulin. In this project, information about the molecular nature of the colchicine binding site and the structure of tubulin will be sought. Knowledge about the structure of the colchicine binding site can be used to discover the naturally occurring substance that may interact with tubulin at the colchicine binding site and therefore be involved in microtubule regulation in he cell. Information about the structure of t ubulin in three dimensions will be useful in understanding what parts of the protein are involved in the contacts that occur in the microtubule and the relationship between these sections and the different regulatory sites on the protein. ***
