Kardos József, Kiss Bence, Micsonai András, Rovó Petra, Menyhárd Dóra K, Kovács János, Váradi Györgyi, Tóth Gábor K, Perczel András
Department of Biochemistry, ‡MTA-ELTE NAP B Neuroimmunology Research Group, and §Department of Anatomy, Cell and Developmental Biology, Institute of Biology Eötvös Loránd University , Pázmány P. sétány 1/C, Budapest, H-1117 Hungary.
J Phys Chem B. 2015 Feb 19;119(7):2946-55. doi: 10.1021/jp5124234. Epub 2015 Feb 6.
The 20 residue long Trp-cage miniprotein is an excellent model for both computational and experimental studies of protein folding and stability. Recently, great attention emerged to study disease-related protein misfolding, aggregation, and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects. It aggregates with ease upon structure destabilization, and thus it is suitable for modeling aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopies, molecular dynamics (MD) simulations, and transmission electron microscopy (TEM) in conjunction with thioflavin-T (ThT) fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation, and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant, which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form that can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.
由20个氨基酸残基组成的色氨酸笼状微蛋白是蛋白质折叠和稳定性计算及实验研究的优秀模型。最近，人们对研究与疾病相关的蛋白质错误折叠、聚集和淀粉样蛋白形成给予了极大关注，目的是揭示其结构和热力学背景。色氨酸笼对环境和结构修饰效应都很敏感。它在结构不稳定时容易聚集，因此适合用于模拟聚集和淀粉样蛋白形成。在此，我们对几种序列修饰和侧链磷酸化的色氨酸笼变体的淀粉样蛋白形成进行了表征。我们结合硫黄素 - T（ThT）荧光测量，应用核磁共振（NMR）、圆二色性（CD）和傅里叶变换红外（FTIR）光谱、分子动力学（MD）模拟以及透射电子显微镜（TEM）来揭示侧链磷酸化的结构后果。我们证明，丝氨酸磷酸化会使天然折叠不稳定，由此产生的高度动态结构会形成具有高分子间β - 结构含量的淀粉样有序聚集体。唯一的例外是D9S(P)变体，它通过形成细纤维遵循另一种聚集过程，呈现Ⅱ型聚脯氨酸螺旋的CD光谱，并且显示出低ThT结合能力。我们为这些色氨酸笼状微蛋白提出了一个复杂的聚集模型。该模型假设存在一种额外的聚集状态，即胶原三螺旋形式，它可能先于淀粉样蛋白形成。单个丝氨酸残基的磷酸化作为一种构象开关，触发聚集，否则由细胞中的激酶介导。我们表明，色氨酸笼状微蛋白确实是具有复合折叠和聚集景观的较大球状系统的现实模型，并有助于我们理解有害蛋白质聚集和淀粉样蛋白形成的基本原理。
