Limited subtilisin cleavage of tubulin results in formation of S-tubulin heterodimer and a 4-kDa carboxylterminal peptide fragment. This carboxyl-terminal domain constitutes an essential site for MAPs interaction and plays a role in modulating the interactions responsible for tubulin self-assembly into microtubules Proc. Natl Acad. Sci. USA 81, 5989; and Biochemistry 23,46751. In the present communication it is shown that addition of the 4-kDa peptide fragment from porcine tubulin to porcine S-tubulin in a molar ratio of about 2: 1 does not affect the assembly of the latter. On the other hand, consistent with previous findings on the binding of the 4-kDa peptide by MAP-2, the peptide inhibited MAP-2-induced tubulin assembly (molar ratio of peptide to tubulin, about 2: 1; peptide to MAP-2, about 30: 1). Comparison of the amino acid composition of the 4-kDa peptide fragment and the C-terminal amino acid residues of S-tubulin with the amino acid sequence of tubulin indicated the subtilisin cleavage site on the tubulin molecule to be between residues G h 4 " and Phe418 of the a-subunit sequence and between Glu407 and Phe4O8 of the B-subunit sequence. The circular dichroism of the 4-kDa fragment in water as solvent is indicative of a molecule with an unordered structure, but when the solvent is changed to a water-trifluoroethanol mixture, the fragment becomes more highly structured.The critical concentration for S-tubulin assembly is not affected by MAPs nor by polylysine, but is decreased by either taxol of dimethylsulfoxide. S-tubulin, with its greater propensity for self-association, has a different conformation from tubulin as shown by a 50% decrease in a-helical content, a more hydrophobic environment of at least some of the tryptophan residues as judged from .fluorimetry, and a greater compaction indicated by flyo = 1.3, as compared to 1.4 for tubulin. The latter point is supported by the observation that the value of the sedimentation coefficient, s20,w = 5.7 S, of the 92-kDa S-tubulin molecule is not significantly different from that of the 100-kDa tubulin, szO,., = 5.8 S.The 100-kDa protein heterodimer tubulin is the subunit protein of microtubules. Tubulin in vitro retains its capacity to self-associate into microtubules, binds nucleotides and interacts with microtubule-associated proteins, MAPs (for reviews see [l -31). Proteolytic dissection of tubulin with several different enzymes has defined structural binding domains for certain small ligands and for MAPs [4--71, revealed accompanying conformational changes resulting from the interactions with these ligands 14, 51 and provided insights into the regulation of tubulin self-association [8]. With respect to tha latter aspect, limited digestion of tubulin with subtilisin results in cleavage at one major site thereby releasing a 4-kDa fragment from the carboxyl-terminal domain of the CI and subunits of tubulin. The cleaved protein heterodimer (S-tubulin), in contrast to tubulin, can self-associate into bundles of microtubules and hooked polymers in the abs...

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A detailed NMR, CD, fluorometry, and molecular modeling study of a novel bradykinin antagonist B-9340, containing a novel amino acid D-Igl (alpha-(2-indanyl)glycine) at position 7, was carried out. The sequence of B-9340 is D-Arg0-Arg1-Pro2-Hyp3-Gly4-Thi5-Ser6-D- Igl7-Oic8-Arg9, where Hyp is hydroxyproline, Thi is beta-(2-thienyl)alanine, and Oic is (3aS,7aS)-octahydroindole-2-carboxylic acid. The CD results exhibit a striking effect of SDS on the spectrum of the BK antagonist, indicating that interaction with the surfactant induces a folded peptide structure. The interaction of this antagonist with phosphatidylinositol was monitored by fluorometry, indicating that the interaction of the peptide with the lipid is cooperative, and gives a Hill coefficient of 2.3. The two-dimensional proton NMR measurements indicate that B-9340 has no stable secondary structure in water solution and contains about 10-15% cis peptide bonds arising from Pro2, Hyp3, and Oic8. In SDS micelles, NMR reveals the existence of two beta-turns based on a number of medium-range connectivities that were useful for molecular modeling. The actual molecular modeling and dynamic runs were performed on B-9340 in an environment consisting of a layer of octyl sulfate anions and water. Ther results indicate that the structure of B-9340 in a micellar environment is characterized by a nonideal betaII-turn comprising residues Pro2 to Thi5, a nonideal betaII'-turn comprising residues Ser6-Arg9, and broad folding in the middle part of the molecule. The structure is stabilized by several hydrogen bonds and by a salt bridge between the guanidine moiety of Arg1 and the carboxyl group of Arg9, whereas the middle part of the peptide is buried in the micelle. The structure is deposited as Brookhaven PDB file 1 BDK.