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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM041117-02
Application #
3299218
Study Section
Metallobiochemistry Study Section (BMT)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
2
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Mississippi Medical Center
Department
Type
Schools of Medicine
DUNS #
928824473
City
Jackson
State
MS
Country
United States
Zip Code
39216
Correia, J J; Gilbert, S P; Moyer, M L et al. (1995) Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341. Biochemistry 34:4898-907
Lobert, S; Frankfurter, A; Correia, J J (1995) Binding of vinblastine to phosphocellulose-purified and alpha beta-class III tubulin: the role of nucleotides and beta-tubulin isotypes. Biochemistry 34:8050-60
Lobert, S; Correia, J J (1994) Method for rapid electrophoretic transfer of isoelectric focusing gels to polyvinylidene difluoride. Electrophoresis 15:930-1
Lobert, S; Isern, N; Hennington, B S et al. (1994) Interaction of tubulin and microtubule proteins with vanadate oligomers. Biochemistry 33:6244-52
Correia, J J; Lipscomb, L D; Dabrowiak, J C et al. (1994) Cleavage of tubulin by vanadate ion. Arch Biochem Biophys 309:94-104
Correia, J J; Lipscomb, L D; Lobert, S (1993) Nondisulfide crosslinking and chemical cleavage of tubulin subunits: pH and temperature dependence. Arch Biochem Biophys 300:105-14
Lobert, S; Hennington, B S; Correia, J J (1993) Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations. Cell Motil Cytoskeleton 25:282-97
Lobert, S; Correia, J J (1992) Subtilisin cleavage of tubulin heterodimers and polymers. Arch Biochem Biophys 296:152-60
Kurland, I J; el-Maghrabi, M R; Correia, J J et al. (1992) Rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Properties of phospho- and dephospho- forms and of two mutants in which Ser32 has been changed by site-directed mutagenesis. J Biol Chem 267:4416-23
Lobert, S; Correia, J J (1992) Antimitotics in cancer chemotherapy. Cancer Nurs 15:22-33

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