Kumar Santosh, Mohanty Subhendu K, Udgaonkar Jayant B
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 560 065, India.
J Mol Biol. 2007 Apr 6;367(4):1186-204. doi: 10.1016/j.jmb.2007.01.039. Epub 2007 Jan 20.
Understanding the heterogeneity of the soluble oligomers and protofibrillar structures that form initially during the process of amyloid fibril formation is a critical aspect of elucidating the mechanism of amyloid fibril formation by proteins. The small protein barstar offers itself as a good model protein for understanding this aspect of amyloid fibril formation, because it forms a stable soluble oligomer, the A form, at low pH, which can transform into protofibrils. The mechanism of formation of protofibrils from soluble oligomer has been studied by multiple structural probes, including binding to the fluorescent dye thioflavin T, circular dichroism and dynamic light scattering, and at different temperatures and different protein concentrations. The kinetics of the increase in any probe signal are single exponential, and the rate measured depends on the structural probe used to monitor the reaction. Fastest is the rate of increase in the mean hydrodynamic radius, which grows from a value of 6 nm for the A form to 20 nm for the protofibril. Slower is the rate of increase in thioflavin T binding capacity, and slowest is the rate of increase in circular dichroism at 216 nm, which occurs at about the same rate as that of the increase in light scattering intensity. The dynamic light scattering measurements suggest that the A form transforms completely into larger size aggregates at an early stage during the aggregation process. It appears that structural changes within the aggregates occur at the late stages of assembly into protofibrils. For all probes, and at all temperatures, no initial lag phase in protofibril growth is observed for protein concentrations in the range of 1 microM to 50 microM. The absence of a lag phase in the increase of any probe signal suggests that aggregation of the A form to protofibrils is not nucleation dependent. In addition, the absence of a lag phase in the increase of light scattering intensity, which changes the slowest, suggests that protofibril formation occurs through more than one pathway. The rate of aggregation increases with increasing protein concentration, but saturates at high concentrations. An analysis of the dependence of the apparent rates of protofibril formation, determined by the four structural probes, indicates that the slowest step during protofibil formation is lateral association of linear aggregates. Conformational conversion occurs concurrently with lateral association, and does so in two steps leading to the creation of thioflavin T binding sites and then to an increase in beta-sheet structure. Overall, the study indicates that growth during protofibril formation occurs step-wise through progressively larger and larger aggregates, via multiple pathways, and finally through lateral association of critical aggregates.
了解淀粉样纤维形成过程中最初形成的可溶性寡聚体和原纤维结构的异质性，是阐明蛋白质淀粉样纤维形成机制的关键环节。小蛋白质巴司他汀是一种很好的模型蛋白，有助于理解淀粉样纤维形成的这一方面，因为它在低pH值下会形成稳定的可溶性寡聚体，即A形式，这种寡聚体可以转化为原纤维。已通过多种结构探针研究了从可溶性寡聚体形成原纤维的机制，这些探针包括与荧光染料硫黄素T结合、圆二色性和动态光散射，研究在不同温度和不同蛋白质浓度下进行。任何探针信号增加的动力学都是单指数的，测量的速率取决于用于监测反应的结构探针。平均流体动力学半径增加的速率最快，从A形式的6纳米增加到原纤维的20纳米。硫黄素T结合能力增加的速率较慢，而在216纳米处圆二色性增加的速率最慢，其发生速率与光散射强度增加的速率大致相同。动态光散射测量表明，在聚集过程的早期，A形式会完全转化为更大尺寸的聚集体。似乎聚集体内的结构变化发生在组装成原纤维的后期。对于所有探针以及所有温度，在1微摩尔至50微摩尔的蛋白质浓度范围内，未观察到原纤维生长的初始滞后阶段。任何探针信号增加过程中不存在滞后阶段，这表明A形式聚集成原纤维不依赖成核。此外，光散射强度增加过程中不存在滞后阶段，而光散射强度变化最慢，这表明原纤维形成通过不止一条途径发生。聚集速率随蛋白质浓度增加而增加，但在高浓度时达到饱和。对由四种结构探针确定的原纤维形成表观速率的依赖性分析表明，原纤维形成过程中最慢的步骤是线性聚集体的横向缔合。构象转换与横向缔合同时发生，并且分两步进行，首先产生硫黄素T结合位点，然后增加β-折叠结构。总体而言，该研究表明，原纤维形成过程中的生长是通过逐渐增大的聚集体逐步进行的，通过多种途径，最终通过关键聚集体的横向缔合实现。
