Abstract：
Many proteins form amyloid fibrils only under conditions when the probability of transition from a native (structured, densely packed) to an intermediate (labile, destabilized) state is increased. It implies the assumption that some structural intermediates are more convenient for amyloid formation than the others. Hence, if a mutation affects the protein folding pathway, one should expect that this mutation could affect the rate of amyloid formation as well. In the current work, we have compared the effects of amino acid substitutions of bovine carbonic anhydrase II on its unfolding pathway and on its ability to form amyloids at acidic pH and an elevated temperature. Wild-type protein and four mutant forms (L78A, L139A, I208A, and M239A) were studied. We analyzed the change of the protein unfolding pathway by the time-resolved fluorescence technique and the process of amyloid formation by thioflavin T fluorescence assay and electron microscopy. It was revealed that I208A substitution accelerates amyloid formation and affects the structure of the late (molten globule-like)-intermediate state of carbonic anhydrase, whereas the other mutations slow down the growth of amyloids and have either no effect on the unfolding pathway (L78A, L139A) or alter the conformational states arising at the early unfolding stage (M239A).
摘要：
许多蛋白质仅在从天然（结构化，密集包装)到中间(不稳定的，不稳定)状态增加。这意味着假设某些结构中间体比其他结构中间体更容易形成淀粉样蛋白。因此，如果突变影响蛋白质折叠途径,人们应该预期这种突变也会影响淀粉样蛋白的形成速度。在目前的工作中,我们已经比较了牛碳酸酐酶II的氨基酸取代对其解折叠途径和在酸性pH和高温下形成淀粉样蛋白的能力的影响。野生型蛋白和四种突变形式(L78A，L139A,I208A,和M239A)进行了研究。我们通过时间分辨荧光技术分析了蛋白质解折叠途径的变化，并通过硫黄素T荧光测定和电子显微镜分析了淀粉样蛋白形成的过程。揭示了I208A取代加速淀粉样蛋白的形成并影响碳酸酐酶的晚期（熔融球样）-中间状态的结构，而其他突变减缓了淀粉样蛋白的生长，并且对解折叠途径没有影响(L78A，L139A）或改变在早期展开阶段出现的构象状态（M239A）。
