Dusa Alexandra, Kaylor Joanna, Edridge Shauna, Bodner Nika, Hong Dong-Pyo, Fink Anthony L
Department of Chemistry and Biochemistry, University of California, Santa Cruz, California 95064, USA.
Biochemistry. 2006 Feb 28;45(8):2752-60. doi: 10.1021/bi051426z.
The aggregation of the presynaptic protein alpha-synuclein is associated with Parkinson's disease (PD). The details of the mechanism of aggregation, as well as the cytotoxic species, are currently not well understood. alpha-Synuclein has four tyrosine and no tryptophan residues. We introduced a tyrosine to tryptophan mutation at position 39 to create an intrinsic fluorescence probe and allow additional characterization of the aggregation process. Y39W alpha-synuclein had similar fibrillation kinetics (2-fold slower), pH-induced conformational changes, and fibril morphology to wild-type alpha-synuclein. In addition to intrinsic Trp fluorescence, acrylamide quenching, fluorescence anisotropy, ANS binding, dynamic light scattering, and FTIR were employed to monitor the kinetics of aggregation. These biophysical probes revealed the significant population of two classes of oligomeric intermediates, one formed during the lag period of fibrillation and the other present at the completion of fibrillation. As expected for a natively unfolded protein, Trp 39 was highly solvent-exposed in the monomer and is solvent-exposed in the two oligomeric intermediates; however, it is partially, but not fully, buried in the fibrils. These observations demonstrate the utility of Trp fluorescence labeled alpha-synuclein and demonstrate the existence of an oligomeric intermediate that exists as a transient reservoir of alpha-synuclein for fibrillation.
突触前蛋白α-突触核蛋白的聚集与帕金森病（PD）相关。目前，聚集机制的细节以及细胞毒性物质尚不清楚。α-突触核蛋白有四个酪氨酸残基，没有色氨酸残基。我们在第39位引入了酪氨酸到色氨酸的突变，以创建一个内在荧光探针，并对聚集过程进行额外的表征。Y39Wα-突触核蛋白的纤维化动力学（慢2倍）、pH诱导的构象变化以及纤维形态与野生型α-突触核蛋白相似。除了内在的色氨酸荧光外，还采用了丙烯酰胺猝灭、荧光各向异性、ANS结合、动态光散射和傅里叶变换红外光谱来监测聚集动力学。这些生物物理探针揭示了两类寡聚中间体的大量存在，一类在纤维化的延迟期形成，另一类在纤维化完成时出现。正如对天然未折叠蛋白的预期，色氨酸39在单体中高度暴露于溶剂中，在两种寡聚中间体中也暴露于溶剂中；然而，它在纤维中部分但未完全被掩埋。这些观察结果证明了色氨酸荧光标记的α-突触核蛋白的实用性，并证明了存在一种寡聚中间体，它作为α-突触核蛋白纤维化的瞬时储存库存在。
