Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant
Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant
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DOI：
10.1021/acs.biochem.0c00436
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发表时间：
2020-10-06
期刊：
影响因子：
2.9
通讯作者：
Kidokoro, Shun-ichi
中科院分区：
文献类型：
--
作者：
Nakamura, Shigeyoshi;Kibria, Md Golam;Kidokoro, Shun-ichi
蛋白质无定形聚集已成为人们关注的焦点，因为它会损害细胞正常工作的能力。在这里，我们评估了三个肽标签，包括一个，三个，和五个连续的异亮氨酸连接在C-末端的一个简化的牛胰胰蛋白酶抑制剂（BPTI）的变体，BPTI-19 A，对热稳定性和寡聚化的圆二色谱和差示扫描量热法的详细影响。所有的BPTI-19 A变体在pH 4.7下表现出可逆的和明显的两态热转变，如BPTI-19 A。五异亮氨酸标记的变体的热转变显示出明显的蛋白质浓度依赖性，其中表观变性温度随着蛋白质浓度的增加而降低。定量分析表明，这种现象起源于可逆低聚（RO）状态在高温下的存在。结果还表明，在所有的蛋白质的天然和单体变性状态之间的热力学稳定性的差异是不稳定的疏水标签，并很好地解释了由于标签的反向疏水效应。RO状态的存在，证实了分析超离心和动态光散射。这表明，五异亮氨酸疏水标签足够强以诱导变性分子之间的分子间疏水接触，导致寡聚化，甚至一个或三个异亮氨酸标签也足够有效以产生分子内疏水接触，从而通过反向疏水效应引起变性。
Protein amorphous aggregation has become the focus of great attention, as it can impair the ability of cells to function properly. Here, we evaluated the effects of three peptide tags, consisting of one, three, and five consecutive isoleucines attached at the C-terminus end of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, BPTI-19A, on the thermal stability and oligomerization by circular dichroism spectrometry and differential scanning calorimetry in detail. All of the BPTI-19A variants exhibited a reversible and apparently two-state thermal transition like BPTI-19A at pH 4.7. The thermal transition of the five-isoleucine-tagged variant showed clear protein-concentration dependence, where the apparent denaturation temperature decreased as the protein concentration increased. Quantitative analysis indicated that this phenomenon originated from the presence of reversibly oligomerized (RO) states at high temperatures. The results also illustrated that the thermodynamic stability difference between the native and the monomeric denatured state in all the proteins was destabilized by the hydrophobic tags and was well explained by the reverse hydrophobic effect due to the tags. The existence of the RO states was confirmed by both analytical ultracentrifugation and dynamic light scattering. This indicated that the five-isoleucine hydrophobic tag is strong enough to induce intermolecular hydrophobic contact among the denatured molecules leading to oligomerization, and even one- or three-isoleucine tags are effective enough to generate intramolecular hydrophobic contact, thus provoking denaturation through the reverse hydrophobic effect.