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Site-directed mutagenesis of beta tubulin's putative GTP-binding domain

Farr, George William
http://rave.ohiolink.edu/etdc/view?acc_num=case1060367517

Abstract Details

1993, Doctor of Philosophy, Case Western Reserve University, Pharmacology.
The cytoskeletal protein tubulin binds guanine nucleotides with high affinity and specificity. GTP binding to the β-subunit requires Mg2+ and stimulates the assembly of tubulin into microtubules. In contrast, GDP does not require Mg2+ and favors disassembly. The structural basis for the competing effects of Mg:GTP and GDP on microtubule assembly remains unclear. In an effort to gain insight into the mechanism of Mg:GTP- and GDP-binding, a site-directed mutagenesis study of β-tubulin's putative GTP-binding domain was initiated. This investigation was performed in four stages: First, the amino acid sequence of β-tubulin was analyzed for homology with other GTP-binding proteins. This analysis revealed three regions of homology, known as the GTP-binding site consensus sequences. As a result, it was proposed that β-tubulin binds GTP in a manner similar to the other GTP-binding proteins, even though these sequences were found in the reverse C- to N-terminal orientation. Second, an in vitro expression system for β-tubulin was developed using rabbit reticulocyte lysate. β-tubulin synthesized in this system was found in three distinct molecular forms: an ∼180 kD non-covalent complex containing β-tubulin, β1; an ∼50 kD fr ee β-subunit, β11; and an ∼100 kD heterodimer containing β-tubulin associated with residual rabbit α-tubulin, β111. These β-polypeptides were shown to be functional by a coassembly assay. The heterodimers were 100% assembly-competent, whereas the 180 kD complex and free β-subunit were ∼70% and ∼30% competent, respectively. Third, a nucleotide-binding assay was developed for the small quantities of β-tubulin synthesized in the in vitro expression system. This assay exploited the observation that the recovery β-tubulin from an anion-exchange column was enhanced by the addition of nucleotide. Fourth, the in vitro expression system and nucleotide-binding assay were used for a site-directed mutagenesis study of β-tubulin's putative GTP-binding domain. Acidic residues were chosen for this study as these residues are known to interact with nucleotide in numerous nucleotide-binding proteins. The results of this mutagenesis study indicate that regions identified by homology with other GTP-binding proteins do not participate, as predicted, in Mg:GTP binding to β-tubulin. Instead, evidence that a region in β-tubulin which has homology with myosin and a number of other ATP-binding proteins interacts with Mg2+ moiety of Mg:GTP was found. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be related to the ATP-binding proteins
Himan Sternlicht (Advisor)
241 p.
Biology, Cell
GTP-binding domain; Mutagenesis, site-directed

Recommended Citations

Citations

  • Farr, G. W. (1993). Site-directed mutagenesis of beta tubulin's putative GTP-binding domain [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1060367517

    APA Style (7th edition)

  • Farr, George. Site-directed mutagenesis of beta tubulin's putative GTP-binding domain. 1993. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1060367517.

    MLA Style (8th edition)

  • Farr, George. "Site-directed mutagenesis of beta tubulin's putative GTP-binding domain." Doctoral dissertation, Case Western Reserve University, 1993. http://rave.ohiolink.edu/etdc/view?acc_num=case1060367517

    Chicago Manual of Style (17th edition)

case1060367517
466
© 1993, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.