Sousa I T, Ruiu L, Lowe C R, Taipa M A
Institute for Biotechnology and Bioengineering (IBB), Centro de Engenharia Biológica e Química, Instituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal.
J Mol Recognit. 2009 Mar-Apr;22(2):83-90. doi: 10.1002/jmr.900.
Cutinase from Fusarium solani pisi is the model-system for a new approach to assess and enhance protein stability based on the use of synthetic triazine-scaffolded affinity ligands as a novel protein-stabilizing tool. The active site of cutinase is excluded from the main surface regions postulated to be involved in early protein's thermal unfolding events. Hence, these regions are suitable targets for binding complementary affinity ligands with a potential stabilizing effect. A random solid-phase combinatorial library of triazine-bisubstituted molecules was screened for binding cutinase by a rapid fluorescence-based method and affinity chromatography. The best binding substituents were combined with those previously selected by screening a rationally designed library. A second-generation solid-phase biased library was designed and synthesized, following a semi-rational methodology. A dual screening of this library enabled the selection of ligands binding cutinase with higher affinity while retaining its functionality. These compounds were utilized for thermostability assessment with adsorbed cutinase at 60 degrees C and pH 8.0. When bound to different types of ligands, the enzyme showed markedly distinct activity retention profiles, with some synthetic affinity ligands displaying a stabilizing effect on cutinase and others a clearly destabilizing effect, when compared with the free enzyme.
来自豌豆镰刀菌的角质酶是一种模型系统，用于基于使用合成三嗪支架亲和配体作为新型蛋白质稳定工具来评估和增强蛋白质稳定性的新方法。角质酶的活性位点位于假定参与蛋白质早期热解折叠事件的主要表面区域之外。因此，这些区域是结合具有潜在稳定作用的互补亲和配体的合适靶点。通过基于快速荧光的方法和亲和色谱法筛选了三嗪双取代分子的随机固相组合文库与角质酶的结合情况。将最佳结合取代基与先前通过筛选合理设计的文库所选择的取代基相结合。按照半理性方法设计并合成了第二代固相偏向文库。对该文库进行双重筛选，能够选择出与角质酶结合亲和力更高同时保留其功能的配体。这些化合物用于在60℃和pH 8.0条件下对吸附的角质酶进行热稳定性评估。当与不同类型的配体结合时，与游离酶相比，该酶显示出明显不同的活性保留曲线，一些合成亲和配体对角质酶有稳定作用，而另一些则有明显的去稳定作用。
