A pressure relaxation study of tubulin oligomer formation

This paper describes a time-resolved X-ray scattering study of microtubule assembly by synchrotron radiation. The method is complementary to light scattering but allows a better distinction between oligomeric and polymeric assembly states. With an improved rapid temperature jump device, it is shown that temperature-induced microtubule assembly is preceded by prenucleation and nucleation events involving oligomers of tubulin, in analogy with earlier results from near-equilibrium temperature scans. In general, the two phases closely overlap, but in certain conditions they can be observed separately. The prenucleation events seen by X-rays can be described as a rapid temperature-dependent equilibrium, with ring oligomers dissociating into smaller oligomers and subunits at elevated temperature. Different solution conditions affect mainly the time lag between the prenucleation and nucleation phases; this in turn determines the apparent magnitude of the prenucleation steps. By contrast, the temperature dependence of the equilibrium between the prenucleation oligomers shows little influence on solution conditions. The results suggest that the ring-forming and tubule-forming assembly modes of tubulin are governed by different interactions between subunits, although they may be based on a pool of similar intermediates.

Section: Discussionsupporting

confidence: 89%

Tubulin oligomers and microtubule assembly studied by time-resolved x-ray scattering: separation of prenucleation and nucleation events

Spann¹,

Renner²,

Bordas³

et al. 1987

“…The isodesmic assembly of tubulin into double rings appears to be very rapid, in all cases more rapid than nucleotide exchange on tubulin. Assuming a plausible range of 1-10 juM-1 s'1 for the rate constant for the tubulin-tubulin association into oligomers, it becomes clear that the formation of rings from oligomers can be completed within 1 s, while the dissociation of GDP from tubulin has a half-time of 6 s (Engelborghs et al, 1980;Brylawski & Caplow, 1983;Melki et al, 1988). Once the equilibrium between double rings and dimers is established, the rapidly labeled oligomers become a smaller fraction of the population.…”

Section: Discussionmentioning

confidence: 99%

Cold depolymerization of microtubules to double rings: geometric stabilization of assemblies

Melki¹,

Carlier²,

Pantaloni³

et al. 1989

177

146

The kinetic pathway of microtubule depolymerization at 0 degrees C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0 degree C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were alpha-beta dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 microM, is much faster than nucleotide exchange on alpha-beta-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on alpha-beta-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the gamma-phosphate in GTP or GDP-Pi.

“…Other investigators have addressed the possibility that rings open and align forming sheets of protofilaments that eventually close into microtubules (Erickson, 1974;Kirschner et al 1974). Karr and Purich (1980) and Engelborghs et al (1980), however, have proposed that rings break down to small oligomers prior to assembly into microtubules. Lee and Timasheff (1977) have suggested that rings are a mode of polymerization independent of microtubule formation.…”

Section: Discussionmentioning

confidence: 99%

GDP state of tubulin: stabilization of double rings

Howard¹,

Timasheff²

1986

116

101

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)