A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases.
A library of fluorescent peptides for exploring the substrate specificities of prolyl isomerases.
复制标题
用于探索脯氨酰异构酶底物特异性的荧光肽库。
作者：
G. Žoldák;T. Aumüller;C. Lücke;Jozef Hritz;C. Oostenbrink;G. Fischer;F. Schmid
为了充分探索脯氨酰异构酶的底物特异性，我们合成了一个由20个四肽组成的文库，这些四肽在氨基末端标记有2-氨基苯甲酰基（Abz）基团，在羧基末端标记有对硝基苯胺（pNA）基团。在该通式为Abz-Ala-Xaa-Pro-Phe-pNA的肽文库中，脯氨酸之前的位置Xaa被所有20个蛋白原氨基酸占据。通过分子动力学模拟和NMR光谱法对肽的构象分析表明，肽中Abz和pNA部分之间的相互距离取决于Xaa-Pro键的异构体状态。在顺式中，但不是在反式中，Abz和pNA部分存在显著的化学位移变化，因为它们的芳环彼此靠近。这种接近性也导致了Abz荧光的强烈淬灭，其与溶剂跳跃结合，用于设计脯氨酰异构酶的灵敏测定。与传统的测定法不同，它不与肽蛋白水解偶联，因此也可用于蛋白酶敏感的脯氨酰异构酶。肽库被用来提供一套完整的P1位点特异性的原型人类成员的三个脯氨酰异构酶家族，FKBP 12，亲环蛋白18，和小蛋白14。在第二个应用程序中，SlyD，蛋白酶敏感的脯氨酰异构酶从大肠杆菌的底物特异性，其特征在于和比较与人FKBP 12以及与同源物从其他细菌。
To fully explore the substrate specificities of prolyl isomerases, we synthesized a library of 20 tetrapeptides that are labeled with a 2-aminobenzoyl (Abz) group at the amino terminus and a p-nitroanilide (pNA) group at the carboxy terminus. In this peptide library of the general formula Abz-Ala-Xaa-Pro-Phe-pNA, the position Xaa before the proline is occupied by all 20 proteinogenic amino acids. A conformational analysis of the peptide by molecular dynamics simulations and by NMR spectroscopy showed that the mutual distance between the Abz and pNA moieties in the peptides depends on the isomeric state of the Xaa-Pro bond. In the cis, but not in the trans form, there are significant chemical shift changes of the Abz and pNA moieties, because their aromatic rings are close to each other. This proximity also leads to a strong quenching of Abz fluorescence, which, in combination with a solvent jump, was used to devise a sensitive assay for prolyl isomerases. Unlike the traditional assay, it is not coupled with peptide proteolysis and thus can be employed for protease-sensitive prolyl isomerases as well. The peptide library was used to provide a complete set of P1-site specificities for prototypic human members of the three prolyl isomerase families, FKBP12, cyclophilin 18, and parvulin 14. In a second application, the substrate specificity of SlyD, a protease-sensitive prolyl isomerase from Escherichia coli, was characterized and compared with that of human FKBP12 as well as with homologues from other bacteria.
影响因子：
2.9
作者：
Wimley, WC;Creamer, TP;White, SH
通讯作者：
White, SH