Limited proteolysis of tubulin: nucleotide stabilizes an active conformation.
Limited proteolysis has been used to examine tubulin structure as related to microtubule assembly. Purified tubulin, freed of exchangeable nucleotide, was digested with low concentrations of chymotrypsin (0.01-0.1% w/w to tubulin) and its polymerization behavior investigated. Chymotryptic proteolysis resulted in a loss of assembly activity with apparent first-order inactivation kinetics. The inactivation rates were dependent on both chymotrypsin concentration and incubation temperature. However, these conditions of proteolysis did not significantly affect tubulin's colchicine binding activity. Polyacrylamide-NaDodSO4 electrophoresis demonstrated the major cleavage fragments of tubulin to be 34 and 17 kilodaltons. Furthermore, amino-terminal analysis showed methionine for the 17-kilodalton fragment and both glutamate and serine for the 34-kilodalton fragment. Microtubular structures formed from chymotryptic tubulin possessed constrictions and had a frayed appearance in the electron microscope; these polymers were composed of both native tubulin and the 34- and 17-kilodalton fragments, suggesting that the loss of microtubule assembly results from tubulin cleavage and the altered interaction of cleavage fragments with uncleaved tubulin subunits. Interestingly, the readdition of GTP prior to proteolysis significantly protected tubulin's assembly capacity, presumably by stabilizing the fragments in an active conformation as indicated by circular dichroism spectra.
D000818:Animals; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006150:Guanine Nucleotides / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011487:Protein Conformation; D012756:Sheep; D014404:Tubulin / Q000378:metabolism*
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D014404:Tubulin; D002918:Chymotrypsin