脯氨酸残基在蛋白质折叠和聚集中起重要作用。我们研究了单个脯氨酸及其组合对人StefinB(stB)的寡聚化和淀粉样蛋白纤维化反应的影响。脯氨酸突变体影响寡聚体在单体之间的分布,二聚体,和四聚体,如尺寸排阻色谱法所示。只有P74S显示出更高的低聚物,让人想起先前报道的stB-Y31变体的P74S的熔融小球。脯氨酸突变体还在不同程度上抑制淀粉样蛋白纤颤反应。在30和37°C时,P74S单突变体的抑制是完全的,两个双突变体(P6LP74S和P74SP79S),和三重突变体P6LP11SP74S。在30°C下,单个突变体P6L完全抑制了反应,而P11S和P79S形成的淀粉样原纤维具有延长的滞后期。P36D没有显示滞后阶段,让人想起下坡聚合模型。除P36D外,在37°C时,P11S,和P79S,P6L和P11SP74S也开始纤维化;然而,通过透射电子显微镜判断,原纤维的产量远低于野生型蛋白。因此,Pro74顺式/反式异构化被证明是关键事件,充当向淀粉样蛋白转变的开关。使用我们以前的成核和生长模型,我们模拟了所有表现出S形纤颤曲线的突变体的动力学。令我们惊讶的是,成核阶段受顺/反异构影响最大,而不是原纤维伸长阶段。
Proline residues play a prominent role in protein folding and aggregation. We investigated the influence of single prolines and their combination on oligomerization and the amyloid fibrillation reaction of human stefin B (stB). The proline mutants influenced the distribution of oligomers between monomers, dimers, and tetramers as shown by the size-exclusion chromatography. Only P74S showed higher oligomers, reminiscent of the molten globule reported previously for the P74S of stB-Y31 variant. The proline mutants also inhibited to various degree the amyloid fibrillation reaction. At 30 and 37 °C, inhibition was complete for the P74S single mutant, two double mutants (P6L P74S and P74S P79S), and for the triple mutant P6L P11S P74S. At 30 °C the single mutant P6L completely inhibited the reaction, while P11S and P79S formed amyloid fibrils with a prolonged lag phase. P36D did not show a lag phase, reminiscent of a downhill polymerization model. At 37 °C in addition to P36D, P11S, and P79S, P6L and P11S P74S also started to fibrillate; however, the yield of the fibrils was much lower than that of the wild-type protein as judged by transmission electron microscopy. Thus, Pro 74 cis/trans isomerization proves to be the key event, acting as a switch toward an amyloid transition. Using our previous model of nucleation and growth, we simulated the kinetics of all the mutants that exhibited sigmoidal fibrillation curves. To our surprise, the nucleation phase was most affected by Pro cis/trans isomerism, rather than the fibril elongation phase.