Department of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany.
Biomacromolecules. 2011 Aug 8;12(8):2988-96. doi: 10.1021/bm200587m. Epub 2011 Jul 13.
The ability to adopt at least two different stable conformations is a common feature of proteins involved in many neurodegenerative diseases. The involved molecules undergo a conformational transition from native, mainly helical states to insoluble amyloid structures that have high β-sheet content. A detailed characterization of the molecular architecture of highly ordered amyloid structures, however, is still challenging. Their intrinsically low solubility and high tendency to aggregate often considerably limits the application of established high-resolution techniques such as NMR and X-ray crystallography. An alternative approach to elucidating the tertiary and quaternary organization within an amyloid fibril is the systematic replacement of residues with amino acids that exhibit special conformational characteristics, such as glycine and proline. Substitutions within the β-sheet-prone sequences of the molecules usually severely affect their ability to form fibrils, whereas incorporation at external loop- and bend-like positions often has only marginal effects. Here we present the characterization of the internal architecture of a de novo designed coiled-coil-based amyloid-forming model peptide by means of a series of systematic single glycine and proline replacements in combination with a set of simple low-resolution methods. The folding and assembly behavior of the substituted peptides was monitored simultaneously using circular dichroism spectroscopy, Thioflavin T fluorescence staining, and transmission electron microscopy. On the basis of the obtained data, we successfully identify characteristic bend and core positions within the peptide sequence and propose a detailed structural model of the internal fibrillar arrangement.
能够采用至少两种不同的稳定构象是涉及许多神经退行性疾病的蛋白质的共同特征。涉及的分子经历构象转变，从天然的、主要是螺旋状态转变为具有高β-折叠含量的不溶性淀粉样结构。然而，对高度有序的淀粉样结构的分子结构的详细表征仍然具有挑战性。它们固有的低溶解度和高聚集倾向常常极大地限制了诸如 NMR 和 X 射线晶体学等既定高分辨率技术的应用。阐明淀粉样纤维内三级和四级组织的替代方法是系统地用表现出特殊构象特征的氨基酸替代残基，例如甘氨酸和脯氨酸。分子中β-折叠倾向序列内的取代通常严重影响其形成纤维的能力，而在外部环和弯曲样位置的掺入通常只有微小的影响。在这里，我们通过一系列系统的单个甘氨酸和脯氨酸取代，并结合一系列简单的低分辨率方法，来描述从头设计的基于卷曲螺旋的淀粉样形成模型肽的内部结构。使用圆二色性光谱、硫黄素 T 荧光染色和透射电子显微镜同时监测取代肽的折叠和组装行为。基于获得的数据，我们成功地确定了肽序列中的特征弯曲和核心位置，并提出了内部纤维排列的详细结构模型。
