Onoda Akira, Yamamoto Hitoshi, Yamada Yusuke, Lee Keonil, Adachi Seiji, Okamura Taka-Aki, Yoshizawa-Kumagaye Kumiko, Nakajima Kiichiro, Kawakami Toru, Aimoto Saburo, Ueyama Norikazu
Department of Macromolecular Science, Graduate School of Science, Osaka University, Toyonaka, Osaka, 560-0043, Japan.
Biopolymers. 2005;80(2-3):233-48. doi: 10.1002/bip.20187.
Aspartic acid protease model peptides Z-Phe-Asp(COOH)-Thr-Gly-Ser-Ala-NHCy (1) and AdCO-Asp(COOH)-Val-Gly-NHBzl (3), and their aspartate anions (NEt4)[Z-Phe-Asp(COO-)-Thr-Gly-Ser-Ala-NHCy] (2) and (NEt4)[AdCO-Asp(COO-)-Val-Gly-NHBzl] (4), having an invariant primary sequence of the Asp-X(Thr,Ser)-Gly fragment, were synthesized and characterized by 1H-NMR, CD, and infrared (IR) spectroscopies. NMR structure analyses indicate that the Asp O(delta) atoms of the aspartate peptide 2 are intramolecularly hydrogen-bonded with Gly, Ser, Ala NH, and Ser OH, supporting the rigid beta-turn-like conformation in acetonitrile solution. The tripeptide in the aspartic acid 3 forms an inverse gamma-turn structure, which is converted to a beta-turn-like conformation because of the formation of the intramolecular NH . . . O- hydrogen bonds with the Asp O(delta) in 4. Such a conformational change is not detected between dipeptides AdCO-Asp(COOH)-Va-NHAd (5) and (NEt4)[AdCO-Asp(COO-)-Val-NHAd] (6). The pK(a) value of side-chain carboxylic acid (5.0) for 3 exhibits a lower shift (0.3 unit) from that of 5 in aqueous polyethyleneglycol lauryl ether micellar solution. NMR structure analyses for 3 in an aqueous micellar solution indicate that the preorganized turn structure, which readily forms the NH . . . O- hydrogen bonds, lowers the pK(a) value and that resulting hydrogen bonds stabilize the rigid conformation in the aspartate anion state. We found that the formation of the NH . . . O- hydrogen bonds involved in the hairpin turn is correlated with the protonation and deprotonation state of the Asp side chain in the conserved amino acid fragments.
天冬氨酸蛋白酶模型肽Z-苯丙氨酸-天冬氨酸（羧基）-苏氨酸-甘氨酸-丝氨酸-丙氨酸-环已基甲酰胺（1）和金刚烷羰基-天冬氨酸（羧基）-缬氨酸-甘氨酸-苄胺（3），以及它们的天冬氨酸阴离子（四乙铵）[Z-苯丙氨酸-天冬氨酸（羧基）-苏氨酸-甘氨酸-丝氨酸-丙氨酸-环已基甲酰胺]（2）和（四乙铵）[金刚烷羰基-天冬氨酸（羧基）-缬氨酸-甘氨酸-苄胺]（4），具有天冬氨酸-X（苏氨酸、丝氨酸）-甘氨酸片段的不变一级序列，通过1H-NMR、圆二色（CD）和红外（IR）光谱进行了合成和表征。核磁共振结构分析表明，天冬氨酸肽2的天冬氨酸O（δ）原子与甘氨酸、丝氨酸、丙氨酸的NH以及丝氨酸的OH形成分子内氢键，支持了在乙腈溶液中类似刚性β-转角的构象。天冬氨酸3中的三肽形成反向γ-转角结构，由于在4中与天冬氨酸O（δ）形成分子内NH…O-氢键，该结构转变为类似β-转角的构象。在二肽金刚烷羰基-天冬氨酸（羧基）-缬氨酸-金刚烷胺（5）和（四乙铵）[金刚烷羰基-天冬氨酸（羧基）-缬氨酸-金刚烷胺]（6）之间未检测到这种构象变化。在聚乙二醇月桂醚胶束水溶液中，3的侧链羧酸的pK（a）值（5.0）相对于5的pK（a）值有较低的偏移（0.3个单位）。在胶束水溶液中对3进行的核磁共振结构分析表明，易于形成NH…O-氢键的预组织转角结构降低了pK（a）值，并且由此产生的氢键稳定了天冬氨酸阴离子状态下的刚性构象。我们发现，发夹转角中涉及的NH…O-氢键的形成与保守氨基酸片段中天冬氨酸侧链的质子化和去质子化状态相关。
