Synthesis of Ca^<2+> -responsive DNA-binding Allosteric Protein

dna结合蛋白被分为几个结构基序。其中之一是两条具有许多基本残基的链的组装，它们通过被称为“亮氨酸拉链”的延伸肽片段彼此靠近。在本研究中，我们使用了环状肽来模拟“亮氨酸拉链”的功能。两个十二肽链连接到环八肽，cyclo(Leu-Sar-Lys(Z)-Sar)_2 （C8KS）和cyclo(Leu-Sar-Lys(CHO)-Sar- leu -Sar- glu (OBzl)-Sar) （C8KE）。两个环肽与Ca^<2+>形成络合物。去除了环肽的保护基团，两条链Boc-Glu-Napala-Leu-Aib-(Lys-Aib-Leu-Aib)_2 -（Napala代表2-萘基丙氨酸）与C8KS （F12-C8KS）结合，Ac-Glu(OMe)- trp - leu - aib -(Lys-Aib-Leu-Aib)_2- leu - aib -Ant （Ant代表一种蒽基衍生物）与C8KE （CH2）结合。CD和荧光光谱分析表明，CH2在缓冲溶液中呈两螺旋链结合的超二级结构。由于以下两个原因，该结构应该是稳定的：1)两个链突出在环骨架的同一侧；2)十二肽呈两亲性的α螺旋构象。当λ DNA加入到alpha缓冲溶液中的CH2中时，Trp残基的荧光被淬灭，而来自蒽基的新信号在更长的波长上出现。因此，CH2与Lambda DNA相互作用，可能将吲哚基和蒽基插入DNA的碱基对中。另一方面，F12-C8KS和单链环肽不与DNA相互作用。因此，CH2的超二级结构应该是与DNA相互作用的关键。有趣的是，Ca^<2+>的存在改变了CH2与DNA的相互作用模式。

DNA-binding proteins are classified into a few structural motifs. One of them is an assembly of two chains with many basic residues, which are brought close with each other by the extended peptide fragment called "Leu zipper". In the present study, we have used cyclic peptides which were expected to mimic function of "Leu zipper". Two chains of dodecapeptides were connected to cyclic octapeptides, cyclo(Leu-Sar-Lys(Z)-Sar)_2 (C8KS) and cyclo(Leu-Sar-Lys(CHO)-Sar-Leu-Sar-Glu(OBzl)-Sar) (C8KE). Both cyclic peptides formed a complex with Ca^<2+>. The protecting groups of the cyclic peptides were removed, and two chains of Boc-Glu-Napala-Leu-Aib-(Lys-Aib-Leu-Aib)_2 - (Napala represents 2-naphthylalanine) were bound to C8KS (F12-C8KS), and Ac-Glu(OMe)-Trp-Leu-Aib-(Lys-Aib-Leu-Aib)_2-and-(Leu-Aib-Glu(ONe)-Aib)_2-Lew-Aib-Ant (Ant represents an anthryl derivative) to C8KE (CH2). CD and fluorescence spectroscopy revealed that CH2 took a super-secondary structure of association of two helical chains in a buffer solution. The structure should be stable due to the following two reasons: i) two chains protrude over the same side of the cyclic skeleton, and ii) the dodecapeptides take an amphiphilic alpha helical conformation.With the addition of Lambda DNA to CH2 in alpha buffer solution, the fluorescence from the Trp residue was quenched, and a new signal from the anthryl group appeared at a longer wavelength. Thus, CH2 interacted with Lambda DNA, probably intercalating the indolyl and anthryl groups into base pairs of the DNA. On the other hand, F12-C8KS and a cyclic peptide having one chain did not interact with DNA. Therefore, the super-secondary structure of CH2 should be critical for the interaction with DNA. Interestingly, the interaction mode of CH2 and DNA was changed by the presence of Ca^<2+>.