Reed J, Hull W E, Ponstingl H, Himes R H
Department of Pathochemistry, German Cancer Research Center, Heidelberg.
Biochemistry. 1992 Dec 1;31(47):11888-95. doi: 10.1021/bi00162a030.
Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.
对主要β-微管蛋白同种型C末端区域的两个肽段（400 - 436和400 - 445）进行了研究，这两个肽段包含与微管相关蛋白2（MAP2）和tau相互作用的关键区域，以确定它们在水溶液中的构象。尽管从理论上看具有形成α-螺旋的高度可能性，但圆二色光谱表明这些肽段主要由无规卷曲和一些反向转角组成。这不受带负电荷侧链的抗衡离子（Ca2 +、Mg2 +）存在的影响，但当通过降低pH使侧链电荷中和时，情况会发生变化；在这些条件下，较长肽段的α-螺旋含量升至25%，C末端截短肽段升至15%。在三氟乙醇存在的情况下，这些肽段也会形成α-螺旋结构，截短肽段再次达到较低的最大百分比。还通过一维和二维核磁共振技术对β（400 - 445）肽段进行了研究。结果表明，在pH 5.6或7的水溶液中，该肽段极具柔性且缺乏规则的二级结构，这与圆二色光谱结果一致。两个肽段均抑制微管相关蛋白刺激的微管蛋白组装，较长肽段的抑制作用约为较小肽段的4倍。在没有微管相关蛋白的情况下，两者均无抑制作用，这表明与该蛋白的相互作用是产生抑制作用所必需的。较长肽段具有更大活性可能是由于该肽段额外的负电荷和/或该肽段在适当条件下形成α-螺旋结构的更大倾向。
