Interaction of tubulin with octyl glucoside and deoxycholate. 2. Protein conformation, binding of colchicine ligands, and microtubule assembly.
The structural change induced by binding of mild detergents to cytoplasmic calf brain tubulin and the effects on the functional properties of this protein have been characterized. Massive binding of octyl glucoside or deoxycholate monomers induces circular dichroism changes indicating a partial alpha-helix to disordered structure transition of tubulin. The protein also becomes more accessible to controlled proteolysis by trypsin, thermolysin, or V8 protease. This is consistent with the looser protein structure proposed in previous binding and hydrodynamic studies [Andreu, J. M., & Muñoz, J. A. (1986) Biochemistry (preceding paper in this issue)]. Micelles of octyl glucoside and deoxycholate bind colchicine and its analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC). This impedes the determination of colchicine binding in the presence of detergents. Both detergents cause a reduction in the number of tubulin equilibrium binding sites for the colchicine site probe MTC. Deoxycholate monomers bind poorly to the tubulin-colchicine complex, but deoxycholate above the critical micelle concentration effectively dissociates the complex. Microtubule assembly in glycerol-containing buffer is inhibited by octyl glucoside, which raises the critical protein concentration. Low concentrations of deoxycholate enhance tubulin polymerization, allowing it to proceed without glycerol. The polymers formed are microtubules, pairwise associated open microtubular sheets, and macrotubules possibly generated by helical folding of the sheets, as indicated by the optical diffraction patterns. Saturation of tubulin with octyl glucoside, followed by full dissociation of the detergent, allowed the recovery of binding to the colchicine site and microtubule assembly, indicating the reversibility of the protein structural change.
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D003078:Colchicine / Q000378:metabolism*; D003840:Deoxycholic Acid / Q000378:metabolism*; D005960:Glucosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism*; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure*; D011485:Protein Binding; D011487:Protein Conformation; D014404:Tubulin / Q000378:metabolism*
D005960:Glucosides; D006027:Glycosides; D014404:Tubulin; D003840:Deoxycholic Acid; C018619:octyl-beta-D-glucoside; D003078:Colchicine