Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recomb inant peptides RL52 alpha 3 and RL33 beta 6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotype s of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. alpha (404-451) and beta(394-445) are monomeric in neutral aqueous solution (as i ndicated by sedimentation equilibrium), and have circular dichroism (CD) sp ectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445 ) with subtilisin, instead of giving extensive degradation, resulted in mai n cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining t he proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terninal helix. Both recombinant pepti des inhibited microtubule assembly, probably due to sequestration of the mi crotubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced m arkedly helical CD spectra in alpha(404-451) and beta(394-445). A substanti al part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional H-1-NMR par ameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alpha H shifts) in 30% TFE permitted to conclude that about 25% of alpha( 404-451) and 40% of beta(394-451) form well-defined helices encompassing re sidues 418-432 and 408-431, respectively, flanked by disordered N- and C-se gments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, T yr422, Tyr425, and Gln426 are well defined in structure calculations from t he NOE distance constraints. The apolar faces of the helix in both alpha an d beta chains share a characteristic sequence of conserved residues Ala,Met (+4),Leu(+7),Tyr(+11). The helical segment of a(404-451) is the same as tha t described in the electron crystallographic model structure of alpha beta- tubulin, while in beta(394-451) it extends for nine residues more, supporti ng the possibility of a functional coil --> helix transition at the C-termi nus of beta-tubulin. These peptides may be employed to construct model comp lexes with microtubule associated protein binding sites.