GDP state of tubulin: stabilization of double rings.
Purified tubulin, with GDP occupying the exchangeable nucleotide binding site, has been examined conformationally and for its ability to self-associate into double rings. The circular dichroism spectrum increased by ca. 10% in negative amplitude between 205 and 225 nm over the spectrum of tubulin in the GTP state, but there were no significant shape changes. This indicates that replacement of GTP by GDP induces tubulin to adopt a more ordered conformation. The sedimentation coefficients of tubulin alpha-beta dimers in the GDP and GTP states were identical, with s20,w = 5.8 S. A sedimentation velocity study of tubulin in the GDP state showed that, in the presence of magnesium ions, this protein undergoes a reversible Gilbert-type self-association. The end product of this reaction was found to be 26 subunit double rings identical with those described by Frigon and Timasheff [(1975) Biochemistry 14, 4567-4599] for a similar polymerization of tubulin in the GTP state. Analysis of the data showed that Tu-GDP has a much stronger propensity for the formation of double rings than Tu-GTP, the corresponding equilibrium with constants for the 26Tu in equilibrium Tu26 being 4.2 X 10(119) M-25 and 2.27 X 10(109) M-25 for Tu-GDP and Tu-GTP, respectively. This leads to Tu-GTP being predominantly in the form of alpha-beta dimers and Tu-GDP in the form of double rings under normal experimental conditions used in the study of microtubule assembly.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D006150:Guanine Nucleotides / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011485:Protein Binding; D011487:Protein Conformation; D013816:Thermodynamics; D014404:Tubulin / Q000378:metabolism*
D006150:Guanine Nucleotides; D046911:Macromolecular Substances; D014404:Tubulin; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate