GDP state of tubulin: stabilization of double rings

Abstract

Purified tubulin, with GDP occupying the exchangeable nucleotide binding site, has been examined conformationally and for its ability to self-associate into double rings. The circular dichroism spectrum increased by ca. 10% in negative amplitude between 205 and 225 nm over the spectrum of tubulin in the GTP state, but there were no significant shape changes. This indicates that replacement of GTP by GDP induces tubulin to adopt a more ordered conformation. The sedimentation coefficients of tubulin alpha-beta dimers in the GDP and GTP states were identical, with s20,w = 5.8 S. A sedimentation velocity study of tubulin in the GDP state showed that, in the presence of magnesium ions, this protein undergoes a reversible Gilbert-type self-association. The end product of this reaction was found to be 26 subunit double rings identical with those described by Frigon and Timasheff [(1975) Biochemistry 14, 4567-4599] for a similar polymerization of tubulin in the GTP state. Analysis of the data showed that Tu-GDP has a much stronger propensity for the formation of double rings than Tu-GTP, the corresponding equilibrium with constants for the 26Tu in equilibrium Tu26 being 4.2 X 10(119) M-25 and 2.27 X 10(109) M-25 for Tu-GDP and Tu-GTP, respectively. This leads to Tu-GTP being predominantly in the form of alpha-beta dimers and Tu-GDP in the form of double rings under normal experimental conditions used in the study of microtubule assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

“…X-ray diffraction datasets were collected at 100K using a MarCCD detector at beam-line ID14 -4 of the European Synchrotron Radiation Facility, Grenoble. Tubulin:RB3-SLD-R and S complexes crystals belong to space group P6 5 . Diffraction data were processed using the HKL package (51).…”

“…Depolymerizing microtubule ends show characteristic curled protofilaments, whereas relatively straight sheets form at growing ends (3,4). GDP-tubulin does not assemble into microtubules, but forms double rings (5), which also form upon microtubule depolymerization (6) and correspond to curved microtubule protofilaments (7,8). The tendency of GDP-tubulin to curve is thought to strain the microtubule lattice, causing disassembly when the terminal cap of GTP-bound tubulin is lost.…”

“…Stathmin (11) is a cellular microtubule inhibitor (12), which forms a tight complex with two ␣␤-tubulin dimers (13), T 2 Stath, 5 binding along their sides and capping the ␣-end (14). T 2 Stath has been shown to be curved by electron microscopy, and it does not polymerize into microtubules (15).…”

“…E-mail: j.m.andreu@ cib.csic.es. 5 The abbreviations used are: T 2 phosphorylation. Proteins of the stathmin family are pivotal microtubule regulators implicated in signal transduction and disease (for reviews, see Refs.…”

A critical assessment of the information processing capabilities of neuronal microtubules using coherent excitations

“…Both monomers can bind a molecule of GTP, but only the β-tubulin will hydrolyze it to GDP. Two different conformational states of the heterodimer are known to depend on this GTP hydrolysis [19] such that hydrolysis causes a small-angle distortion between the original center-to-center line joining the monomers, releasing 0.42 eV of energy per molecule in free tubulin and a few times less that amount when embedded in a MT [20]. On average, an αβ-heterodimer has dimensions of 46 × 80 × 65 Å and is a polar molecule with its positive end near the β-subunit [21].…”