Carboxy-terminal amino acid sequence of α-tubulin from porcine brain

Ponstingl, Herwig; Little, Melvyn; Krauhs, Erika; Kempf, Tore

doi:10.1038/282423a0

Cited by 52 publications

(35 citation statements)

References 20 publications

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“…The longer helix of the b-tubulin model peptide suggests an extension in the protein, supporting the possibility of a functional coil r helix transition at the C-terminal zone. This is consistent with an earlier proposal based on the mixed helix-coil prediction~Ponstingl et al., 1979;Fig. 1!. Following this helix, the 19 and 14 C-terminal residues of the respective a-and b-tubulin model peptide are observed to be disordered by NMR.…”

Section: Comparison Of the Conformation Of The C-terminal Zones In Tfsupporting

confidence: 94%

Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides

et al. 1999

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We have investigated the solution conformation of the functionally relevant C-terminal extremes of a-and b-tubulin, employing the model recombinant peptides RL52a3 and RL33b6, which correspond to the amino acid sequences 404-451~end! and 394-445~end! of the main vertebrate isotypes of a-and b-tubulin, respectively, and synthetic peptides with the a-tubulin~430-443! and b-tubulin~412-431! internal sequences. a~404-451! and b~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 b~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 b-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol TFE!-induced markedly helical CD spectra in a~404-451! and b~394-445!. A substantial part of the helicity of b~394-445! was found to be in the CD spectrum of the shorter peptide b~412-431! with TFE. Two-dimensional 1 H-NMR parameters~nonsequential nuclear Overhauser effects~NOE! and conformational CaH shifts! in 30% TFE permitted to conclude that about 25% of a~404-451! and 40% of b~394-451! form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N-and C-segments. The side chains of b~394-451! residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both a and b chains share a characteristic sequence of conserved residues Ala,Met~ϩ4!,Leu~ϩ7!,Tyr~ϩ11!. The helical segment of a~404-451! is the same as that described in the electron crystallographic model structure of ab-tubulin, while in b~394-451! it extends for nine residues more, supporting the possibility of a functional coil r helix transition at the C-terminus of b-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.

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Section: Comparison Of the Conformation Of The C-terminal Zones In Tfsupporting

confidence: 94%

Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides

et al. 1999

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“…All major helix potentials reside in the COOH-terminal half of the chain around residues 275-291 and the three helices at the COOH-terminus already reported in an earlier paper (17), residues 383-403, 413-435, and 440-450. Major /8-sheet regions are expected at positions 49-94 (five strands), (three strands), and 223-239 and 340-378 (four strands).…”

Section: Asn-leu-asn-arg-leu-ile-gly-gln-ile-val-ser-ser-ile-thr-ala-supporting

confidence: 61%

“…A striking feature of the sequence is the COOH-terminal region, which we already have discussed in detail (17). The last 66 residues are entirely devoid of asparagine, glutamine, threonine, cysteine, proline, and isoleucine, and the last 40 positions have 47% acidic side chains, 16 glutamic and three aspartic, rendering this segment one of the most acidic known.…”

Section: Resultsmentioning

confidence: 92%

Complete amino acid sequence of alpha-tubulin from porcine brain.

Ponstingl¹,

Krauhs²,

Little³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

273

155

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The amino acid sequence of a-tubulin from porcine brain was determined by automated and manual Edman degradation of eight sets of overlapping peptides. It comprises 450 residues plus a COOH-terminal tyrosine that is present only in 15% of the material. A region of 40 residues at the COOH-terminus is highly acidic, mainly due to 16 glutamyl residues. This high concentration ofnegative charge suggests a region for binding cations. At least six positions, most of them around position 270, are occupied by two amino acid residues each. Several of these exchange sites were assigned to specific peptides by analysis of the purified corresponding fragments. These data indicate four a-tubulins in porcine brain. Although a-tubulin on the whole is unrelated to other proteins, there are regions that can be correlated to sequences of the myosin head, to actin, to tropomyosin, and to troponins C and T.Tubulins occur in all eukaryotic cells as the constituents of microtubules, which participate in cell division, intracellular transport and secretion processes, ciliary and flagellar movement, morphogenesis, and cell orientation. Tubulins from widely differing species and cell types appear to be remarkably similar regarding composition, molecular weight, binding of cytostatic and psychopharmacological drugs, immunological crossreactivity, and capacity to copolymerize. Yet even within one cell, there are several types of microtubules that have differing stabilities and assemble into distinct organelles at various times. Knowledge of the primary structure should clarify whether there is just one tubulin for all functions or whether there exists a family of similar proteins. It will also facilitate mapping of binding sites for various ligands, production of antibodies to well-defined antigenic sites, matching of protein structure with that of messengers and genes, and investigation of functionally defective tubulin mutants. Comparison of the structure with those of known proteins may give hints for experiments regarding tubulin function.Tubulin in solution is assumed to exist as a heterodimer of two chains, a and f, each with a molecular weight4of 50,000, and very similar amino acid compositions. Yet functional differences have been reported. For example, only a-tubulin (from blood platelets) binds cyclic AMP (1) and only /3-tubulin binds exchangeable GTP (2). Here we present the sequence of the a-chain from porcine brain and report on the general strategy used. MATERIALS AND METHODSWe have purified tubulin from porcine brain by a modification of the methods used by Eipper (3) and by Luduena et al. (4). The 100,000 X g brain supernatant in 0.05 M sodium pyrophosphate buffer (pH 7.0) was incubated with 0.1 mM colchicine for 15 min at 37°C before chromatography on DEAE-cellulose with a linear gradient of 0.1-0.3 M sodium chloride. Tubulin was identified by the fluorescence of its complex with colchicine (5). The preparation was reduced, alkylated with iodoacetic acid, and assayed for protein impurities by disc gel electrophore...

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“…The interaction between spermine and the ␣Tub410C peptide was then examined in detail using two-dimensional 1 Fig. 4C, which shows that two regions of the ␣Tub410C peptide are influenced by the interaction with spermine: amino acid residues 430 -432 and 444 -451.…”

Section: Resultsmentioning

confidence: 99%

“…Microtubules consist mainly of ␣␤-tubulin heterodimers organized head-to-tail into protofilaments whose parallel self-association gives rise to microtubules (1)(2)(3)(4). ␣-and ␤-tubulin monomers have each a molecular mass of 50 kDa and are organized in three domains, namely the N-terminal domain (amino acid residues 1-205) involved in nucleotide binding, the intermediate domain (amino acid residues 206 -384), and the C-terminal domain (amino acid residue 385 to the C terminus) (5).…”

mentioning

confidence: 99%

The C Terminus of Tubulin, a Versatile Partner for Cationic Molecules

Lefèvre¹,

Chernov

Joshi

et al. 2011

Journal of Biological Chemistry

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The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau Microtubules are involved in a number of critical cellular processes, such as the determination of cell shape, chromosome segregation, intracellular transport of vesicles and organelles, and cell migration. Microtubules consist mainly of ␣␤-tubulin heterodimers organized head-to-tail into protofilaments whose parallel self-association gives rise to microtubules (1-4). ␣-and ␤-tubulin monomers have each a molecular mass of 50 kDa and are organized in three domains, namely the N-terminal domain (amino acid residues 1-205) involved in nucleotide binding, the intermediate domain (amino acid residues 206 -384), and the C-terminal domain (amino acid residue 385 to the C terminus) (5). The C-terminal domain of tubulin represents a critical part of the binding site of different tubulin/microtubules partners, such as MAPs, 4 which are major regulators of microtubule dynamics (6 -8), or polycations, which promote tubulin assembly in vitro in different polymeric forms (9). The C-terminal domain comprises a highly negatively charged tail of about 20 amino acid residues (named herein the C-terminal tail (CTT)), which protrudes from the surface of microtubules. In agreement with its participation in the regulation of microtubule assembly through interactions with partners, the CTT is also the most divergent part of tubulin, and variations among tubulin isotypes (10) may explain the modulation of the dynamics of microtubule assembly in specific tissues or cytoplasmic regions.Different structure information has been obtained regarding the C-terminal domain of tubulin by using either fulllength tubulin or peptide fragments. Electron crystallography of zinc-induced tubulin sheets showed the presence of two anti-parallel ␣-helices (helix H11 (amino acid residues 385-397) and helix H12 (amino acid residues 418 -433)) lying at the outer surface of tubulin. The regions corresponding to the CTTs of either ␣-or ␤-tubulin were however not observed, probably due to the flexibility of this part of the protein (5). These observations were confirmed by x-ray diffraction analyses of crystal complexes formed between tubulin and the RB3-stathmin-like domain (11-13). Other structural data were obtained with peptides from the C-terminal region of tubulin studied either when free in solution or in interaction with different partners. NMR structure investigations on ␣-and ␤-tubulin C-terminal peptides showed that both ␣ (residues 404 -451) and ␤ (residues 394 -445) peptides have no defined secondary structure in aqueous solution but contain a well □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental 4 The abbreviations used are: MAP, microtubule-associated protein; ␣Tub410C, amino acid residues 410 -451 from ␣1a-tubulin; CTT, tubulin C-terminal tail; ITC, isothermal titration ...

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Carboxy-terminal amino acid sequence of α-tubulin from porcine brain

Cited by 52 publications

References 20 publications

Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides

Helicity of α(404–451) and β(394–445) tubulin C‐terminal recombinant peptides

Complete amino acid sequence of alpha-tubulin from porcine brain.

The C Terminus of Tubulin, a Versatile Partner for Cationic Molecules

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