Department of Pharmaceutical Science, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198-6025, USA.
J Phys Condens Matter. 2012 Apr 25;24(16):164205. doi: 10.1088/0953-8984/24/16/164205. Epub 2012 Mar 30.
Self-assembly of misfolded proteins into ordered fibrillar structures is a fundamental property of a wide range of proteins and peptides. This property is also linked with the development of various neurodegenerative diseases such as Alzheimer's and Parkinson's. Environmental conditions modulate the misfolding and aggregation processes. We used a peptide, CGNNQQNY, from yeast prion protein Sup35, as a model system to address effects of environmental conditions on aggregate formation. The GNNQQNY peptide self-assembles in fibrils with structural features that are similar to amyloidogenic proteins. Atomic force microscopy (AFM) and thioflavin T (ThT) fluorescence assay were employed to follow the aggregation process at various pHs and ionic strengths. We also used single molecule AFM force spectroscopy to probe interactions between the peptides under various conditions. The ThT fluorescence data showed that the peptide aggregates fast at pH values approaching the peptide isoelectric point (pI = 5.3) and the kinetics is 10 times slower at acidic pH (pH 2.0), suggesting that electrostatic interactions contribute to the peptide self-assembly into aggregates. This hypothesis was tested by experiments performed at low (11 mM) and high (150 mM) ionic strengths. Indeed, the aggregation lag time measured at pH 2 at low ionic strength (11 mM) is 195 h, whereas the lag time decreases ~5 times when the ionic strength is increased to 150 mM. At conditions close to the pI value, pH 5.6, the aggregation lag time is 12 ± 6 h under low ionic strength, and there is minimal change to the lag time at 150 mM NaCl. The ionic strength also influences the morphology of aggregates visualized with AFM. In pH 2.0 and at high ionic strength, the aggregates are twofold taller than those formed at low ionic strength. In parallel, AFM force spectroscopy studies revealed minimal contribution of electrostatics to dissociation of transient peptide dimers.
错误折叠的蛋白质自行装配成有序的纤维状结构，是广泛的蛋白质和肽的基本特性。这一特性也与阿尔茨海默病和帕金森病等各种神经退行性疾病的发展有关。环境条件调节错误折叠和聚集过程。我们使用酵母朊病毒 Sup35 的 CGNNQQNY 肽作为模型系统，以解决环境条件对聚集形成的影响。GNNQQNY 肽在纤维中自行组装，其结构特征与淀粉样蛋白形成蛋白相似。原子力显微镜（AFM）和硫黄素 T（ThT）荧光分析用于在各种 pH 值和离子强度下跟踪聚合过程。我们还使用单分子 AFM 力谱在各种条件下探测肽之间的相互作用。ThT 荧光数据表明，在接近肽等电点（pI=5.3）的 pH 值下，肽迅速聚集，在酸性 pH（pH2.0）下的动力学速度慢 10 倍，这表明静电相互作用有助于肽自组装成聚集体。通过在低（11 mM）和高（150 mM）离子强度下进行的实验验证了这一假设。实际上，在低离子强度（11 mM）下 pH 2 时测量的聚集滞后时间为 195 h，而当离子强度增加到 150 mM 时，滞后时间减少约 5 倍。在接近 pI 值（pH5.6）的条件下，在低离子强度下，聚集滞后时间为 12±6 h，而在 150 mM NaCl 下，滞后时间几乎没有变化。离子强度也会影响 AFM 可视化的聚集体形态。在 pH2.0 和高离子强度下，聚集体比在低离子强度下形成的聚集体高两倍。同时，AFM 力谱研究表明，静电对瞬态肽二聚体的解离几乎没有贡献。
