Jain Shweta, Udgaonkar Jayant B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, Bangalore 560065, India.
J Mol Biol. 2008 Oct 24;382(5):1228-41. doi: 10.1016/j.jmb.2008.07.052. Epub 2008 Jul 26.
The full-length mouse prion protein, moPrP, is shown to form worm-like amyloid fibrils at pH 2 in the presence of 0.15 M NaCl, in a slow process that is accelerated at higher temperatures. Upon reduction in pH to 2, native moPrP transforms into a mixture of soluble beta-rich oligomers and alpha-rich monomers, which exist in a slow, concentration-dependent equilibrium with each other. It is shown that only the beta-rich oligomers and not the alpha-rich monomers, can form worm-like amyloid fibrils. The mechanism of formation of the worm-like amyloid fibrils from the beta-rich oligomers has been studied with four different physical probes over a range of temperatures and over a range of protein concentrations. The observed rate of fibrillation is the same, whether measured by changes in ellipticity at 216 nm, in thioflavin fluorescence upon binding, or in the mean hydrodynamic radius. The observed rate is significantly slower when monitored by total scattering intensity, suggesting that lateral association of the worm-like fibrils occurs after they form. The activation energy for worm-like fibril formation was determined to be 129 kJ/mol. The observed rate of fibrillation increases with an increase in protein concentration, but saturates at protein concentrations above 50 microM. The dependence of the observed rate of fibrillation on protein concentration suggests that aggregate growth is rate-limiting at low protein concentration and that conformational change, which is independent of protein concentration, becomes rate-limiting at higher protein concentrations. Hence, fibril formation by moPrP occurs in at least two separate steps. Longer but fewer worm-like fibrils are seen to form at low protein concentration, and shorter but more worm-like fibrils are seen to form at higher protein concentrations. This observation suggests that the beta-rich oligomers grow progressively in size to form critical higher order-oligomers from which the worm-like amyloid fibrils then form.
全长小鼠朊病毒蛋白（moPrP）在pH 2、0.15 M NaCl存在的条件下，会形成蠕虫状淀粉样纤维，这是一个缓慢的过程，在较高温度下会加速。当pH值降至2时，天然moPrP会转变为富含β-片层的可溶性寡聚体和富含α-螺旋的单体的混合物，它们以缓慢的、浓度依赖性的平衡状态相互存在。研究表明，只有富含β-片层的寡聚体而非富含α-螺旋的单体能够形成蠕虫状淀粉样纤维。在一系列温度和蛋白质浓度范围内，使用四种不同的物理探针研究了富含β-片层的寡聚体形成蠕虫状淀粉样纤维的机制。通过216 nm处椭圆率的变化、结合时硫黄素荧光的变化或平均流体动力学半径来测量，观察到的纤维化速率是相同的。当通过总散射强度监测时，观察到的速率明显较慢，这表明蠕虫状纤维在形成后会发生横向聚集。确定蠕虫状纤维形成的活化能为129 kJ/mol。观察到的纤维化速率随蛋白质浓度的增加而增加，但在蛋白质浓度高于50 μM时达到饱和。观察到的纤维化速率对蛋白质浓度的依赖性表明，在低蛋白质浓度下聚集体生长是限速步骤，而在高蛋白质浓度下与蛋白质浓度无关的构象变化成为限速步骤。因此，moPrP形成纤维至少发生在两个独立的步骤中。在低蛋白质浓度下会形成更长但数量更少的蠕虫状纤维，而在高蛋白质浓度下会形成更短但数量更多的蠕虫状纤维。这一观察结果表明，富含β-片层的寡聚体尺寸逐渐增大，形成关键的高阶寡聚体，然后从中形成蠕虫状淀粉样纤维。
