Zoldák Gabriel, Aumüller Tobias, Lücke Christian, Hritz Jozef, Oostenbrink Chris, Fischer Gunter, Schmid Franz X
Laboratorium für Biochemie und Bayreuther Zentrum für Molekulare Biowissenschaften, Universität Bayreuth, D-95440 Bayreuth, Germany.
Biochemistry. 2009 Nov 3;48(43):10423-36. doi: 10.1021/bi9014242.
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.
为了全面探究脯氨酰异构酶的底物特异性，我们合成了一个包含20种四肽的文库，这些四肽在氨基末端用2-氨基苯甲酰（Abz）基团标记，在羧基末端用对硝基苯胺（pNA）基团标记。在这个通式为Abz-Ala-Xaa-Pro-Phe-pNA的肽文库中，脯氨酸之前的Xaa位置被所有20种蛋白质ogenic氨基酸占据。通过分子动力学模拟和核磁共振光谱对该肽进行的构象分析表明，肽中Abz和pNA部分之间的相互距离取决于Xaa-Pro键的异构状态。在顺式而非反式构象中，Abz和pNA部分有显著的化学位移变化，因为它们的芳香环彼此靠近。这种接近还导致Abz荧光的强烈猝灭，结合溶剂跳跃，被用于设计一种灵敏的脯氨酰异构酶检测方法。与传统检测方法不同，它不与肽的蛋白水解偶联，因此也可用于对蛋白酶敏感的脯氨酰异构酶。该肽文库用于为脯氨酰异构酶三个家族的典型人类成员FKBP12、亲环蛋白18和小泛素相关修饰蛋白14提供一套完整的P1位点特异性。在第二个应用中，对来自大肠杆菌的一种对蛋白酶敏感的脯氨酰异构酶SlyD的底物特异性进行了表征，并与人类FKBP12以及其他细菌的同源物进行了比较。
