Dipartimento di Scienze Fisiche ed Astronomiche, University of Palermo, Via Archirafi 36, 90123 Palermo, Italy.
Arch Biochem Biophys. 2011 Apr 1;508(1):13-24. doi: 10.1016/j.abb.2011.01.024. Epub 2011 Feb 15.
We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering, Circular Dichroism (CD), infrared spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Changes in protein secondary structures turn out to be the driving mechanism of the observed aggregation and they progress in parallel with the growth of Thioflavin T emission intensity and scattering signal. This concurrent behavior suggests a mutual stabilization of elongated protofibril-like structures and of protein conformational and structural changes, which lead to a more rigid and ordered structures. Our results give new insights on BSA self-assembly process in alkaline conditions clearly providing new pieces of evidences of the interplay of several and interconnected mechanisms occurring on different time and length scales.
我们报告了一项关于模型蛋白牛血清白蛋白（BSA）的实验研究，旨在阐明完全折叠的球状蛋白如何通过不同的聚集途径形成淀粉样纤维或无定形聚集体。我们观察到在远离蛋白质等电点的 pH 值下，热诱导形成纤维状结构。静电斥力的增加导致蛋白质失稳，并改变分子间和分子内相互作用，导致由分子间β-sheet 稳定的纤维状聚集物的生长。通过荧光技术、光散射、圆二色性（CD）、红外光谱（FTIR）和原子力显微镜（AFM）研究了聚集动力学。蛋白质二级结构的变化被证明是观察到的聚集的驱动力，它与硫黄素 T 发射强度和散射信号的增长平行进行。这种并发行为表明，伸长的原纤维状结构和蛋白质构象和结构变化之间存在相互稳定作用，这导致了更刚性和有序的结构。我们的结果为 BSA 在碱性条件下的自组装过程提供了新的见解，清楚地提供了关于在不同时间和长度尺度上发生的几种相互关联的机制相互作用的新证据。
