Amorphous protein aggregation monitored using fluorescence self-quenching
Amorphous protein aggregation monitored using fluorescence self-quenching
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DOI：
10.1002/1873-3468.12439
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发表时间：
2016-10-01
期刊：
影响因子：
3.5
通讯作者：
Kuroda, Yutaka
中科院分区：
文献类型：
--
作者：
Jozawa, Hiroki;Kabir, Md. Golam;Kuroda, Yutaka
对无定形蛋白质聚集的生物物理理解可以显著影响生物技术的各个领域。在这里，我们报告的时间依赖性盐诱导形成的无定形聚集监测荧光自淬灭和比较结果与传统的方法检测蛋白质聚集[静态光散射（LS）和动态光散射（DLS）]。作为模型蛋白，我们使用了牛胰胰蛋白酶抑制剂（BPTI）变体，其在C末端由两个甘氨酸（C2 G）延伸，以及三种变体，其中由五个丝氨酸（C5 S）、丙氨酸（C5 A）或天冬氨酸（C5 D）制成的三种类型的溶解度控制肽标签（SCP标签）被添加到C2 G的C末端。所有变体都具有天然样BPTI结构和胰蛋白酶抑制活性，但不同的溶解度由SCP标签控制。使用与BPTI的赖氨酸缀合的NHS-黄绿素（FAM）标记BPTI，并且我们测量了在加入NaCl后发生的荧光强度的变化。添加盐后，所有FAM-BPTI的荧光几乎立即下降，尽管程度不同，并在10分钟后保持恒定，持续24小时或更长时间。另一方面，LS和DLS信号的变化依赖于标签的类型。即C2 G的LS和DLS信号迅速变化，C5 S和C5 A标记的FAM-BPTIs信号从10 min到24 h缓慢增加，而C5 D的信号保持不变。这些观察结果表明存在至少一个中间步骤，增加蛋白质-蛋白质相互作用产生“分子缩合”相。根据该模型，C2 G将迅速从“缩合物”转变为聚集体，而C5 S和C5 A标记的FAM-BPTI将缓慢地转变，并且可溶性C5 D标记的变体将保持在分子缩合状态。
Biophysical understanding of amorphous protein aggregation can significantly impact diverse area of biotechnology. Here, we report the time dependent salt-induced formation of amorphous aggregation as monitored by fluorescence self-quenching and compare the results with conventional methods for detecting protein aggregation [static light scattering (LS) and dynamic light scattering (DLS)]. As a model protein, we used a bovine pancreatic trypsin inhibitor (BPTI) variant extended by two glycines (C2G) at its C terminus, and three variants where three types of Solubility Controlling Peptide tags (SCP tags) made of five serines (C5S), alanines (C5A) or aspartic acids (C5D) were added to the C terminus of C2G. All variants have a native-like BPTI structure and trypsin inhibitory activity, but different solubilities controlled by the SCP tags. The BPTIs were labeled using NHS-Fluorescein (FAM) conjugated to BPTI's lysines, and we measured the changes in fluorescence intensity occurring upon the addition of NaCl. The fluorescence of all FAM-BPTIs decreased almost immediately, albeit to a different extent, upon addition of salt and became constant after 10 min for 24 h or more. On the other hand, LS and DLS signal changes were dependent on the type of tags. Namely, C2G's LS and DLS signals changed immediately, the signals of C5S and C5A tagged FAM-BPTIs increased slowly from 10 min to 24 h, and those of C5D remained constant. These observations indicated the presence of at least one intermediate step, with increased protein-protein interaction yielding a 'molecular condensation' phase. According to this model, C2G would rapidly turn from 'condensates' to aggregates, whereas C5S and C5A tagged FAM-BPTIs would do so slowly, and the soluble C5D tagged variant would remain in the molecular condensation state.