Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation.
Influence of the stability of a fused protein and its distance to the amyloidogenic segment on fibril formation.
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DOI：
10.1371/journal.pone.0015436
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发表时间：
2010-11-23
期刊：
影响因子：
3.7
通讯作者：
Schwarz E
中科院分区：
文献类型：
--
作者：
Buttstedt A;Winter R;Sackewitz M;Hause G;Schmid FX;Schwarz E
天然蛋白质转化为淀粉样纤维是不可逆的，因此很难通过热力学分析来研究构象稳定性和纤维化的相互依赖性。在这里，我们解决了这个问题，融合淀粉样蛋白的聚丙氨酸片段来自核聚（A）结合蛋白PABPN 1的N-末端结构域与一个研究充分，可逆展开蛋白，CspB从枯草芽孢杆菌。早期的研究表明，CspB可以保持其折叠结构的原纤维，当它是从淀粉样蛋白的部分分开的一个很长的连接。当CspB与淀粉样蛋白生成区段直接融合时，它展开，因为其N-末端链区域整合到纤维核心中，如蛋白酶作图实验所示。CspB和淀粉样蛋白生成片段之间3或16个残基的间隔区不足以防止CspB结构的丧失。由于CspB的低热力学稳定性（ΔG D = 12.4kJ/mol）可能是CspB解折叠和整合到纤维中的原因，因此研究了具有增强的热力学稳定性（ΔG D = 26.9kJ/mol）的CspB突变体的融合。 这种强烈稳定的CspB保持折叠并防止所有融合体中的原纤维形成。我们的数据表明，连接的，独立结构的蛋白质结构域的构象稳定性可以控制原纤维的形成。
Conversion of native proteins into amyloid fibrils is irreversible and therefore it is difficult to study the interdependence of conformational stability and fibrillation by thermodynamic analyses. Here we approached this problem by fusing amyloidogenic poly-alanine segments derived from the N-terminal domain of the nuclear poly (A) binding protein PABPN1 with a well studied, reversibly unfolding protein, CspB from Bacillus subtilis. Earlier studies had indicated that CspB could maintain its folded structure in fibrils, when it was separated from the amyloidogenic segment by a long linker. When CspB is directly fused with the amyloidogenic segment, it unfolds because its N-terminal chain region becomes integrated into the fibrillar core, as shown by protease mapping experiments. Spacers of either 3 or 16 residues between CspB and the amyloidogenic segment were not sufficient to prevent this loss of CspB structure. Since the low thermodynamic stability of CspB (ΔG D = 12.4 kJ/mol) might be responsible for unfolding and integration of CspB into fibrils, fusions with a CspB mutant with enhanced thermodynamic stability (ΔG D = 26.9 kJ/mol) were studied. This strongly stabilized CspB remained folded and prevented fibril formation in all fusions. Our data show that the conformational stability of a linked, independently structured protein domain can control fibril formation.
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