Fassina G, Consonni R, Zetta L, Cassani G
Protein Engineering Unit, TECNOGEN S.c.p.A., Milan, Italy.
Int J Pept Protein Res. 1992 Jun;39(6):540-8. doi: 10.1111/j.1399-3011.1992.tb00286.x.
Molecular recognition between Big Endothelin (Big ET) and a computer generated peptide hydropathically complementary to Big ET[16-29] sequence has been studied by analytical high performance liquid affinity chromatography (HPLAC), circular dichroism (CD) and nuclear magnetic resonance (NMR) experiments. Specific binding was observed between solid support immobilized complementary peptide and Big ET[1-38], [1-32], and [16-32], but not with Big ET fragments [1-21], [16-21], [22-32], and [22-38], obtained by chymotrypsin proteolytic degradation. Selectivity in the recognition process was clearly demonstrated by the ability of complementary peptide affinity column to purify the Big ET molecule from complex peptide mixtures, even when present in very low concentrations. Similar selectivity was evidenced with the Big ET fragment [16-32], [NH2-HLDIIWVNTPEHIVPYG-COOH] containing the entire hydropathically complementary sequence. Binding was followed by marked spectroscopic changes, as monitored by circular dichroism and one- and two-dimensional nuclear magnetic resonance experiments. The NMR spectra of the complementary peptides 1:1 mixture showed variations in the chemical shifts of proton resonances in several residues, both in the main chain (amide protons) and in the side chains (aliphatic and aromatic protons). These data support the hypothesis of a multilocalized type of interaction between complementary peptides, where many residues along the peptide chains participate in co-operative stabilizing contacts in the forming complex.
通过分析型高效液相亲和色谱（HPLAC）、圆二色光谱（CD）和核磁共振（NMR）实验，研究了大内皮素（Big ET）与计算机生成的与Big ET[16 - 29]序列具有亲水性互补的肽之间的分子识别。观察到固定在固体支持物上的互补肽与Big ET[1 - 38]、[1 - 32]和[16 - 32]之间存在特异性结合，但与通过胰凝乳蛋白酶蛋白水解降解获得的Big ET片段[1 - 21]、[16 - 21]、[22 - 32]和[22 - 38]不存在特异性结合。互补肽亲和柱能够从复杂的肽混合物中纯化Big ET分子，即使其浓度非常低，这清楚地证明了识别过程中的选择性。含有完整亲水性互补序列的Big ET片段[16 - 32]，[NH2 - HLDIIWVNTPEHIVPYG - COOH]也证明了类似的选择性。通过圆二色光谱以及一维和二维核磁共振实验监测，结合后伴随着明显的光谱变化。互补肽1:1混合物的NMR谱显示，几个残基的质子共振化学位移发生了变化，包括主链（酰胺质子）和侧链（脂肪族和芳香族质子）。这些数据支持了互补肽之间存在多定位相互作用类型的假设，即沿着肽链的许多残基参与形成复合物时的协同稳定接触。
