The long range goal of the proposed research is a detailed molecular understanding of the mechanism of GTP hydrolysis and its relation to the dynamics of microtubule assembly/disassembly. We will perform extensive thermodynamic and kinetic studies of the binding of guanine nucleotides and their analogues to the tubulin heterodimer over a wide range of temperatures and assembly conditions. Microtubule assembly is nucleated by a GTP-Mg-tubulin complex and requires the presence of GTP and Mg2+ (or other divalent or trivalent cations). Therefore, competitive binding studies will be undertaken in the presence of other metals, especially Mn2+ and Al3+, and over a wide range of ratios of GDP/GTP to explore the energetics of metal and nucleotide binding. The metal dependence of the binding of nonhydrolyzable (GMPPCP and GMPPNP) and thio-analogues of guanine nucleotides will be studied to explore the steroselective requirements at the exchangeable-catalytic, nucleotide binding site of tubulin. The rate of exchange of GTP for GDP as a function of metal concentration will be explored by the change in intrinsic protein fluorescence upon binding of S6-GTP or S6-GDP. These studies will be extended to microtubules by monitoring the rate of GTP hydrolysis as a function of the concentration of various divalent cations and ratios of GDP/GTP. The hydrolysis of GTP is presumably followed by the release of MgPO4 and the generation of dynamically unstable microtubules. The rate of dissociation of Pi and the dissociation and exchange of various metals from microtubules will be investigated. Changes in the secondary structure and intrinsic fluorescent properties of tubulin or bound fluorescent nucleotides upon nucleotide exchange will be studied. The cycle of microtubule assembly/disassembly is regulated by the events subsequent to GTP hydrolysis: a conformational change in GDP-tubulin triggers disassembly and a divalent cation-GTP complex exchanges for GDP initiating the reassembly of the """"""""recharged"""""""" subunits into growing populations of microtubules. A knowledge of the energetics of divalent cation-nucleotide binding and exchange, and the mechanism of GTP hydrolysis is central to an understanding of the temporal dynamics of this important cytoplasmic system.
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