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. 2002 Jun;13(6):2120-31.
doi: 10.1091/mbc.e01-10-0089.

Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source

Michael Caplow  1 , Lanette Fee
Affiliations

Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source

Michael Caplow et al. Mol Biol Cell. 2002 Jun.

Abstract

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.

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Figures

Figure 1
Figure 1
Surface plasmon resonance analysis of biotin-tubulin binding to strepavidin and subsequent dimer dissociation as a result of dilution. (A) The plasmon resonance signal was increased by 954 RU during a 10-min flow of biotin-tubulin in Pi buffer with 12 mM Mg. The almost instantaneous 3000 RU signal change at the start and finish of the flow of the tubulin resulted from a difference in refractive index of the tubulin solution and the buffer. (B) Flow of tubulin- and nucleotide-free buffer resulted in a 445 RU signal decrease; the curve corresponds to a rate constant 14.72 × 10−5 s−1. A rate constant equal to 12.35 × 10−5 s−1 was determined from a Guggenheim plot of the data.
Figure 2
Figure 2
Rate of dissociation of tubulin-GTP (A) and of tubulin without E-site nucleotide (B). (A) After biotin-tubulin-GTP in BRB buffer with 10 μM GTP was bound to produce a 2027 RU signal, the chip was washed with protein-free BRB buffer with 20 μM GTP. (A) The rate constant was 1.94 × 10−5 s−1 and the signal decrease was 1081 RU. (B) The initial binding of tubulin in BRB without nucleotide gave a signal increase of 1720 RU, and the change during flow of tubulin-free BRB buffer without nucleotide was 523 RU. It is expected that because of dimer dissociation 6% of the signal was lost during the binding (calculated by assuming that binding and dissociation are consecutive first-order processes).
Figure 3
Figure 3
Molecular-weight dependence of protein elution from a Superdex 200 column in BRB buffer. Calibration was with thyroglobulin, apoferritin, amylase, bovine serum albumin, egg albumin, and carbonic anhydrase, left to right, respectively.
Figure 4
Figure 4
Stability of tubulin-GDP. Tubulin-GDP in BRB buffer, 0.2 μM, was chromatographed on Superdex immediately after dilution (▵) and after incubation for 10 (●) and for 22 h (+). The peaks were at 13.73, 13.69, and 13.73 ml, respectively, in these reactions. Virtually identical results were obtained in three experiments.
Figure 5
Figure 5
Role of Mg in stabilizing tubulin-GDP. Tubulin-GDP, 0.2 μM, was incubated in BRB containing 10 mM EDTA for 1.5 (A), 5 (B), and 12 h (C) before chromatography. At 1.5 h the main peak was at 13.69 ml (103 kDa).
Figure 6
Figure 6
Stability of 0.2 μM tubulin-GDP analyzed with an immunochemical assay. Samples were chromatographed after 2 (A) and after 11.5 (B) h. The signal strength varied because of assay conditions.
Figure 7
Figure 7
Chromatography of 2 nM tubulin-GDP as a function of time. Samples were chromatographed immediately after dilution (A; apparent MW 113 kDa), after 3 h (B, apparent MW 105 kDa), after 5 h (C, apparent MW 119 kDa), and after 19 h (D, apparent MW 107 kDa). The signal strength varied because of assay conditions.
Figure 8
Figure 8
Tubulin dissociation at low concentrations. Tubulin-GDP at 0.2 (A), 0.04 (B), and 0.02 nM (C) was chromatographed after incubation for 3–4 h. A major signal was at ∼13.56 ml (109 kDa) in A; at ∼13.56 ml (109 kDa) and 15.16 ml (51 kDa) in B; and at ∼13.74 ml (100 kDa) and 14.54 ml (69 kDa) in C.
Figure 9
Figure 9
Kinetics for dissociation of tubulin-GDP after dilution to 2 nM. The rate was calculated from Eq. 3.
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