Institute of Biophysics, National Research Council of Italy, via U. La Malfa 153, I-90146 Palermo, Italy.
J Phys Chem B. 2012 Oct 11;116(40):12078-87. doi: 10.1021/jp303430g. Epub 2012 Oct 2.
The role of intermolecular interaction in fibril-forming protein solutions and its relation with molecular conformation is a crucial aspect for the control and inhibition of amyloid structures. Here, we study the fibril formation and the protein-protein interactions of lysozyme at acidic pH and low ionic strength. The amyloid formation occurs after a long lag time and is preceded by the formation of oligomers, which seems to be off-pathway with respect to fibrillation. By measuring the osmotic isothermal compressibility and the collective diffusion coefficient of lysozyme in solution, we observe that the monomeric solution is kept in a thermodynamically metastable state by strong electrostatic repulsion, even in denaturing conditions. The measured repulsive interaction between monomers is satisfactorily accounted for by classical polyelectrolyte theory. Further, we observe a slow conformational change involving both secondary and tertiary structure, which drives the proteins toward a more hydrophobic conformation. Denatured proteins are driven out of metastability through conformational substates, which are kinetically populated and experience a lower activation energy for fibril formation. Thus, our results highlight the role of electrostatic repulsion, which hinders the aggregation of partially denatured proteins and operates as a gatekeeper favoring the association of those monomers whose conformation is capable of forming amyloid structure.
分子间相互作用在纤维状蛋白溶液中的作用及其与分子构象的关系是控制和抑制淀粉样结构的关键方面。在这里，我们研究了溶菌酶在酸性 pH 值和低离子强度下的纤维形成和蛋白-蛋白相互作用。淀粉样形成发生在很长的滞后时间之后，并且在寡聚体形成之前发生，这似乎与纤维形成不同。通过测量溶液中溶菌酶的渗透压等温压缩系数和集体扩散系数，我们观察到即使在变性条件下，强静电排斥也使单体溶液处于热力学亚稳状态。单体之间的测量到的排斥相互作用可以用经典的聚电解质理论很好地解释。此外，我们观察到涉及二级和三级结构的缓慢构象变化，这促使蛋白质向更疏水的构象转变。变性蛋白通过构象亚稳态被逐出亚稳状态，这些亚稳态在动力学上是多相的，并且形成纤维所需的激活能较低。因此，我们的结果强调了静电排斥的作用，它阻碍了部分变性蛋白的聚集，并作为一种门控机制，有利于那些构象能够形成淀粉样结构的单体的结合。
