Abstract
The self-assembly of tubulin devoid of microtubule-associated proteins (MAPs) has been studied using a MES buffer containing dimethyl sulfoxide (Me2SO). Between 6% and 12% v/v Me2SO, the tubulin forms polymers which resemble microtubules in their morphology and chemical properties. These Me2SO microtubules, like normal microtubules, require GTP for assembly and are sensitive to cold, calcium ions, colchicine, and hydrostatic pressure. The polymerization shows a critical concentration which is dependent on the concentration of Me2SO. 8% Me2SO was found to be the optimum concentration for microtubule assembly. In these conditions, a linear Van t'Hoff plot is obtained, with delta H0/kJ . mol-1 = 26.5 over the range of 10-35 degrees C, and delta S0/J . K-1 . mol-1 = 186, in contrast to the assembly with MAPS or glycerol. The kinetics of polymerization shows that the apparent stoichiometry coefficient of nucleation has the value of 2. Ultracentrifugation analysis shows that there are no oligomers present at low temperatures in the absence of free nucleotide, while in identical conditions, tubulin with MAPs does form oligomers. Although the solvent conditions used supported propagation of assembly, nucleation was found to be very dependent on the transiently locally high Me2SO concentrations formed when Me2SO was added to initiate assembly. It is concluded that Me2SO preferentially stabilizes the lateral interactions.
Highlights
The relation between mass concentrationof polytween 6% and 12%v/v MeZSO, the tubulin forms poly- mer and turbidity is linear in MezSO, while it deviates strongly mers which resemble microtubuilnestheir morphology from linearity inglycerol due to nonideality [5]
Hydrostatic pressure.The polymerization shows a crit- In the presence of MAPs, the Van t'Hoff plot of the propical concentration which is dependent on the concentration of MezSO.8%MezSOwas found to be the optimum concentration for microtubule assembIlyn.these conditions, a linear Van t'Hoff plot is obtained, with AHo/kJ*mol" = 26.5overtherange 10-35 "C, and ASo/J=K"-mol" = 186, in contrast to the assembly with MAPS or glycerol
The kinetics of polymerization agation equilibrium is biphasic. This is attributed to theffect of the MAPs [8]
Summary
Jane Robinson$ and Yves Engelborghs From the Laboratory of Chemical and Biological Dynamics, Katholieke Universiteit te Leuven, Celestijnenlaan 200 D,B-3030Leuuen, Belgium. Hydrostatic pressure.The polymerization shows a crit- In the presence of MAPs, the Van t'Hoff plot of the propical concentration which is dependent on the concentration of MezSO.8%MezSOwas found to be the optimum concentration for microtubule assembIlyn.these conditions, a linear Van t'Hoff plot is obtained, with AHo/kJ*mol" = 26.5overtherange 10-35 "C, and ASo/J=K"-mol" = 186, in contrast to the assembly with MAPS or glycerol. MezSO is known to stabilize microtubules formed in shows that the apparent stoichiometry coefficient of the presence of MAPs [9]. These effects ofMeZSO are of nucleationhas the valueof 2. The solvent conditions used supported propagation of assembly, nucleatiown as found
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