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 recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes 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 indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra 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 main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix.

Effect of 2'-OH acetylation on the bioactivity and conformation of 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol. A NMR-fluorescence microscopy study.

The relationship between conformation, 2'-OH acetylation, and bioactivity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present the structure of taxol with the 7-OH group esterified with the N-(4'-fluoresceincarbonyl)-L-alanine group and with the 2'-OH group free (taxoid 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d6/D2O (3/7), showing that both taxoids adopt a similar collapsed conformation in which the hydrophobic groups 2-O-benzoyl, 3'-phenyl and 4-O-acetyl are in close proximity, with the fluorescein group displaying unrestricted motion.

Stability of Escherichia coli phosphoenolpyruvate carboxykinase against urea-induced unfolding and ligand effects.

The urea-induced unfolding at pH 7.5 of Escherichia coli phosphoenolpyruvate (P-pyruvate) carboxykinase was studied by monitoring the enzyme activity, intrinsic protein fluorescence, circular dichroism spectra, and 1-anilino-8-naphthalenesulfonate binding. These studies were performed in the absence and presence of substrates and ligands.

Fluorescent taxoids as probes of the microtubule cytoskeleton.

Microtubules are specifically and efficiently visualized with the new fluorescent taxoids 7-O-[N-(4'-fluoresceincarbonyl)-L-alanyl]taxol (FLUTAX) and 7-O-[N-(4'-tetramethylrhodaminecarbonyl)-L-alanyl]taxol (ROTAX). Similarly to taxol, FLUTAX and ROTAX are able to drive inactive GDP-liganded tubulin into microtubule assembly. One molecule of FLUTAX binds per alphabeta-tubulin dimer assembled, competing with taxol for the same microtubule binding site with an eightfold smaller relative affinity.

Tubulin secondary structure analysis, limited proteolysis sites, and homology to FtsZ.

The far-ultraviolet circular dichroism spectrum of the alpha beta-tubulin dimer analyzed by six different methods indicates an average content of approximately 33% alpha helix, 21% beta sheet, and 45% other secondary structure. Deconvolution of Fourier transform infrared spectra indicates 24% sheet, 37% (maximum) helix, and 38% (minimum) other structure. Separate alignments of 75 alpha-tubulin, 106 beta-tubulin, and 14 gamma-tubulin sequences and 12 sequences of the bacterial cell division protein FtsZ have been employed to predict their secondary structures with the multiple-sequence method PHD [Rost, B.