亚纳米单分子定位技术研究化学修饰对蛋白-膜相互作用的干预

Alpha-synuclein (AS) is a small (140-residue,14.5 kDa), soluble presynaptic protein that is highly conserved in vertebrates and has been implicated in Parkinson's disease, which is characterized by aggregation of AS into oligomers. Interaction between AS and lipids in the neuronal cell membrane has been proposed to be an important step in the process of oligomerization and cytotoxicity. Effects of post-translational modifications (phosphorylation, nitration, oxidation, methylation, acetylation, ubiquitination, and so on) on protein secondary structure and susceptibility to conformational changes can be dramatic. Even a small subpopulation of modified AS could have a substantial impact on kinetics and product distribution. The molecular mechanisms and the potential sequence of events involved in the formation of oligomers are unclear. Here we apply a newly developed technique, surface-induced fluorescence attenuation (SIFA) to study the intervention of protein-membrane interactions by chemical modifications. This sub-nanometer single-molecule localization technique aims to proble configurational modifications in AS and their effects on its conversion into disease-related forms. Our studies would have great impact on clarifying the links between AS and Parkinson's disease.

Alpha-synuclein (AS) 是由140个氨基酸组成的高丰度前突触蛋白，是帕金森病人大脑内Lewy小体的重要成分。AS在生物膜上聚集初期形成的可溶性寡聚物的毒性要强于多聚物，探讨影响AS聚集的动态过程意义重大。AS的氨基酸序列中存在众多能被翻译后修饰的位点, 不同修饰影响其与磷脂的结合、自身的聚集以及毒性作用。本项目计划发展并优化在亚纳米精度实时测量蛋白质与生物膜精细相互作用的单分子荧光技术，测量AS在膜上的折叠、插膜和聚集，通过单分子水平的新实验检验文献中提出的多种分子模型，研究化学修饰对AS折叠和聚集的干预与调控，甄别化学修饰与非化学修饰的AS的动态聚集的不同点，为建立化学修饰对AS初始聚集的干预和调控的模型提供定量科学依据。

膜蛋白与膜的相互作用是蛋白质发挥生物学功能的重要途径，测量膜蛋白的动态过程对定量理解膜蛋白的干预和调控具有重要意义。细胞膜的尺寸仅有数纳米，因此发展高精度精密测量技术是至关重要的。本项目以发展亚纳米精度的单分子荧光定位方法为基础，并运用该方法研究神经性疾病关键蛋白Alpha-synuclein (AS) 、细胞凋亡关键蛋白tBid等膜蛋白，对其膜蛋白变异、小分子调控等动态过程进行精密定位测量，澄清了此前有争议的问题，并提出了新的分子模型，为理解致病机理和治疗策略提供科学依据。

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