Rashno Fatemeh, Khajeh Khosro, Dabirmanesh Bahareh, Sajedi Reza H, Chiti Fabrizio
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Biomedical, Experimental and Clinical Sciences, Section of Biochemistry, University of Florence, Viale Morgagni 50, Florence, Italy.
Protein Eng Des Sel. 2018 Nov 1;31(11):419-426. doi: 10.1093/protein/gzz003.
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.
此前，从假单胞菌属来源纯化的一种脂肪酶显示，在没有可检测到的延迟期的情况下，它能非常迅速地形成淀粉样纤维结构。在此过程中，尿素展开的酶遇到接近生理状态的介质，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域（APR）。确定了两个聚集倾向最高的区域。这两个区域是51 - 57区和160 - 172区，因为在所有四种预测算法中都发现了它们。选择了脂肪酶的两个突变体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）和动态光散射评估它们的聚集倾向。虽然I52E脂肪酶形成的聚集体能够与淀粉样染料结合，呈现典型的β - 折叠结构和无定形/纤维状形态，但F171E突变体形成的聚集体显示出ThT结合减少、光散射降低、β - 折叠结构含量较低以及通过TEM成像显示聚集体存在较少。这些数据表明，序列160 - 172区域是该蛋白质的一个APR区域，并提示了旨在提高该蛋白质溶解度的策略。
