A synchrotron X-ray scattering characterization of purified tubulin and of its expansion induced by mild detergent binding.
This report presents a synchrotron radiation X-ray scattering characterization of calf brain tubulin purified by the modified Weisenberg procedure. The results show that under nonassembly conditions (i.e., in 10 mM sodium phosphate and 0.1 mM GTP, pH 7, buffer) these preparations consist of a uniform population of molecules with a radius of gyration of 3.1 +/- 0.1 nm, which can be interpreted as arising from the native alpha-beta heterodimer. The uniformity in the population persists even at unusually high concentrations of protein. Binding of colchicine or substitution of GTP by GDP does not induce, within the statistical accuracy and resolution range of our measurements, any significant structural modification in soluble tubulin. In assembly buffer [i.e., 10 mM sodium phosphate, 6 mM magnesium chloride, 1 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, 1 mM GTP, and 3.4 M glycerol, pH 6.5], these preparations readily assemble into microtubules upon increasing the temperature from 4 to 37 degrees C. Binding of nondenaturing amphiphiles to soluble tubulin provides a simplified model for tubulin-membrane interactions. The X-ray scattering data show that the radius of gyration of tubulin progressively increases upon binding of the mild detergent sodium deoxycholate, reaching a maximum value of 4.3 +/- 0.1 nm at detergent saturation. The relative increase in the radius of gyration coincides within experimental error with the previously determined relative increase in the frictional coefficient [Andreu, J.M., & Muñoz, J.A. (1986) Biochemistry 25, 5220-5230]. Analysis of these observations suggests that the effect of detergent binding is to induce an isotropic swelling of the protein structure.
Manuel Andreu
J
J
Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
Garcia de Ancos
J
J
Starling
D
D
Hodgkinson
J L
JL
Bordas
J
J
D000818:Animals; D001921:Brain / Q000378:metabolism; D002021:Buffers; D002417:Cattle; D003840:Deoxycholic Acid / Q000494:pharmacology; D007700:Kinetics; D046911:Macromolecular Substances; D008433:Mathematics; D010315:Particle Accelerators; D011487:Protein Conformation; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*; D014961:X-Ray Diffraction / Q000379:methods
D002021:Buffers; D046911:Macromolecular Substances; D014404:Tubulin; D003840:Deoxycholic Acid