INVESTIGATION OF THE MECHANISM OF AMYLOID FIBRIL FORMATION FROM PRESSURE EXPERIMENTS

1. 我们利用压力跳质子核磁共振波谱发现，在施加压力的情况下，缺乏二硫化物的母鸡溶菌酶的淀粉样原纤维被解离成单体。在压力下解离是完全可逆的；也就是说，在降低压力的情况下，释放的单体蛋白重新结合成原纤维。利用色氨酸荧光在变压和原子力显微镜下对上述现象进行了详细的研究。通过对实验结果的分析，确定了二硫化物缺失母鸡溶菌酶的缔合和解离是通过线性聚合机制进行的，即单体蛋白分别附着在原原纤维的一端和从原原纤维的一端分离。解离速率与压力密切相关。压力依赖性分析表明，解离的活化体积为负（ΔV^<o【双正】>=-50.5±1.60 ml mol^<-1>），活化压缩率也为负。这些结果使我们得出结论，原原纤维是一个高体积状态，部分水化导致激活状态解离。在0 ~ 37℃的温度范围内，利用色氨酸荧光监测，研究了转甲状腺素野生型和V30M突变体的热力学稳定性与压力的关系。外推到1 bar的稳定性表明，V30M突变体在整个研究温度范围内稳定性较差。结果证实了我们之前从高压核磁共振得出的结论，即V30M突变体淀粉样纤维的形成与其热力学不稳定性有关。

1. We have found, by using pressure-jump proton NMR spectroscopy, that the amyloid protofibrils of disulfide-deficient hen lysozyme are dissociated into monomeric species by applying pressure. The dissociation is fully reversible with pressure ; namely, upon lowering pressure, the freed monomeric proteins restart association into protofibrils.2. The above phenomenon is studied in detail using Trp fluorescence under variable pressure and atomic force microscopy. The analysis of the experimental results have established that the association and dissociation of the disulfide-deficient hen lysozyme takes place by the linear polymerization mechanism, in which the monomeric protein is attached and detached to and from one end of the protofibril, respectively. The dissociation rate is strongly pressure-dependent. The analysis of the pressure dependence shows that the activation volume for the dissociation is negative (ΔV^<o【double plus】>=-50.5±1.60 ml mol^<-1>) and that the activation compressibility is also negative. These results have led us to conclude that the protofibril is a high-volume state, partial hydration of which leads to the activated state for dissociation.3. Thermodynamic stability of the wild-type and V30M mutant of transthyretin is studied as a function of pressure using Trp fluorescence as monitor in the temperature range between 0 and 37℃. The stability extrapolated to 1 bar indicates that V30M mutant is less stable in the entire temperature range studied. The results confirms our earlier conclusion from high pressure NMR that the amyloid fiber formation of V30M mutant is correlated to its thermodynamic instability.