Design of a peptide hormone: synthesis and characterization of a model peptide with calcitonin-like activity
Design of a peptide hormone: synthesis and characterization of a model peptide with calcitonin-like activity
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肽激素的设计：具有降钙素样活性的模型肽的合成和表征
DOI：
10.1021/ja00350a064
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发表时间：
1983
影响因子：
15
通讯作者：
E. Kaiser
中科院分区：
文献类型：
--
作者：
G. Moe;Richard J. Miller;E. Kaiser
很明显，残基分离到圆柱形片段相对的亲水面和疏水面是所有这些分子的共同特征。此外，残基8至22区域中的相对两亲性α螺旋特征与降钙素的生物活性之间似乎存在相关性。基于这些观察，我们提出了降钙素结构模型，其中存在三个结构区域：涉及半胱氨酸残基1和7之间的二硫桥的“环”，从残基8开始到位置22结束的两亲螺旋片段，以及从残基23到残基32的亲水无规卷曲序列。为了测试两亲螺旋结构对降钙素生物活性的重要性，我们合成了一种模型肽（MCT-I，图 1），并使用鲑鱼降钙素 I (SCT-I)（天然存在的降钙素活性最高且对酶促降解最稳定的降钙素）作为比较标准，表征了其化学和生物学特性。在残基8至22的区域中，模型肽被设计为与任何天然降钙素具有尽可能小的序列同源性，同时保持与SCT-1相同的电荷平衡和疏水性与亲水性。此外，选择具有高螺旋形成潜力的氨基酸来构成MCT-I的螺旋区。 2 因此，选择亮氨酸作为疏水残基，选择谷氨酰胺作为中性亲水残基，选择赖氨酸作为碱性亲水残基。在MCT-1分子的剩余部分中，残基1-7对应于人降钙素的残基1-7的序列，并且残基23-32对应于SCT-1的等效区域。由于色氨酸在未来的荧光研究中可能有用，因此将其放置在螺旋区域疏水面的中间。 MCT-I 通过固相法合成，并通过 CM-Seph-adex C-25 上的离子交换色谱法和 Waters C18 半制备柱上的反相 HPLC 纯化至均质。 3 当肽从 Waters C18 反相柱中使用 20% 至 50% CHjCN 梯度作为洗脱溶剂洗脱时，在 230 nm 处观察到单个对称峰，并根据 5.5 Mhc1 水解后的氨基酸分析来判断该肽是纯的。氨基酸分析如下：Arg，1.11(1)；天冬氨酸，2.01 (2)；半胱氨酸，2.09 (2)；谷氨酸，5.00 (5)；甘氨酸，3.02 (3)；亮氨酸，7.14 (7)；赖氨酸，2.94 (3)；专业版，1.63 (2)；系列，1.61 (2)；苏尔，3.5 (4)。 MCT-1 和 SCT-I 的圆二色性 (CD) 光谱
it was readily apparent that the segregation of residues into opposing hydrophilic and hydrophobic faces of cylindrical segments was a feature common to allof them. Furthermore, there appeared to be a correlation between the relative amphiphilic a-helical character in the region from residues 8 to 22 and thebiological activities of the calcitonins. On the basis of these observations, we propose a model for calcitonin structure in which there are three structural regions: a “loop” involving the disulfide bridge between cysteine residues 1 and 7, an amphiphilic-helical seg-ment beginning at residue 8 and ending at position 22, and a hydrophilic random coil sequence from residue 23 to residue 32. To test the importance of amphiphilic-helical structure to the biological activity of the calcitonins, we have synthesized a model peptide (MCT-I, Figure 1) and characterized its chemical and biological properties, using salmon calcitonin I (SCT-I), the naturally occurring calcitonin most active and most stable to enzymatic degradation, as the standard for comparison. In the region from residues 8 to 22 the model peptide was designed to have as little sequence homology as possible to any of the natural calcitonins, while retaining the same balance of charge and hydrophobicity to hydrophilicity as SCT-I. In addition, amino acids having a high helix-forming potential were chosen toconstitute the helical region of MCT-I. 2 Thus, leucines were selected as the hydrophobic residues, glutamines as neutral hydrophilic residues, and lysines as basic hydrophilic residues. In the re-mainder of the MCT-I molecule, residues 1-7 correspond to the sequence from residues 1 to 7 of human calcitonin, and residues 23-32 correspond to the equivalent region of SCT-I. Because of its possible usefulness in future fluorescence studies, Trp was placed in the middle of the hydrophobic face of the helical region. MCT-I was synthesized by the solid-phase method and purified to homogeneity by ion-exchangechromatography on CM-Seph-adex C-25 and reverse-phase HPLC on a Waters C18 semipreparative column. 3 The peptide was judged tobe pure on the basis of the observation at 230 nm of a single symmetrical peak when the peptide was eluted from a Waters C18 reverse-phase column using a gradient from 20% to 50% CHjCN as the eluting solvent and from its amino acid analysis after hydrolysiswith 5.5 Mhc1. The amino acid analysis was as follows: Arg, 1.11 (1); Asp, 2.01 (2); Cys, 2.09 (2); Glu, 5.00 (5); Gly, 3.02 (3); Leu, 7.14 (7); Lys, 2.94 (3); Pro, 1.63 (2); Ser, 1.61 (2); Thr, 3.5 (4). The circular dichroism (CD) spectra of MCT-1 and SCT-I from