Previously, a lipase purified from a Pseudomonas source showed to form amyloid fibril structure very rapidly in the absence of a detectable lag phase. In this process the urea-unfolded enzyme encounters a medium close to physiological, but is unable to fold and, therefore, the main driving force of aggregation lies in the sequence of the protein and in its aggregation-promoting regions (APRs). Two regions with the highest propensity to aggregate were identified. These were Regions 51–57 and 160–172 as they were found with all four prediction algorithms. Two mutants of lipase, F171E and I52E, were selected and their propensity to aggregate was evaluated using thioflavin T (ThT), Congo red binding, circular dichroism, transmission electron microscopy (TEM) and dynamic light scattering. While I52E lipase formed aggregates that were capable of amyloid dye binding, showed a typical β-sheet structure and amorphous/fibrillar morphology, the aggregates formed by the F171E mutant indicated diminished ThT binding, lower light scattering, a smaller content of β-sheet structure and a lower presence of aggregates by TEM imaging. These data indicate that the region of the Sequence 160–172 is an APR region of this protein and lead to the suggestion of strategies aimed at promoting the solubility of this protein.

中文翻译：

---

洞悉假单胞菌属脂肪酶的聚集。使用诱变：通过采用α-螺旋结构来保护易于聚集的区域

以前，从假单胞菌来源纯化的脂肪酶显示在没有可检测到的滞后相的情况下非常迅速地形成淀粉样蛋白原纤维结构。在此过程中，未折叠的尿素酶会遇到接近生理的介质，但无法折叠，因此，聚集的主要驱动力在于蛋白质的序列及其聚集促进区（APR）。确定了两个聚集倾向最高的区域。这是在所有四种预测算法中发现的51-57区和160-172区。选择了两个脂肪酶突变体F171E和I52E，并使用硫黄素T（ThT），刚果红结合，圆二色性，透射电子显微镜（TEM）和动态光散射评估了它们的聚集倾向。尽管I52E脂肪酶形成了能够与淀粉样蛋白染料结合的聚集体，表现出典型的β-折叠结构和无定形/原纤维形态，但F171E突变体形成的聚集体显示ThT结合力降低，光散射较低，β-折叠结构含量较小通过TEM成像可以降低聚集体的含量。这些数据表明，序列160–172的区域是该蛋白质的APR区域，并导致了旨在提高该蛋白质溶解度的策略的建议。