脂质体包裹荧光受体方法研究a-突触核蛋白在磷脂膜上的结构和动态特征

Investigation of structure and dynamics of a-synuclein on membrane by quenchers-in-a-liposome fluorescence resonance energy transfer method

α-突触核蛋白(α-synuclein,α-syn)在帕金森病的发病机理中起着关键的作用,因而近年来受到了越来越广泛的关注.α-syn在膜上的动力学过程对理解其功能至关重要.本文使用基于脂质体的单分子荧光衰减方法——LipoFRET,首次对较高浓度下α-syn与磷脂膜的相互作用的动态过程进行了单分子层面上的研究.研究发现,在溶液中α-syn浓度升高时,其中央NAC区域可离开磷脂膜表面进入水相中;而N端部分位于膜表面内的位置变浅,并有更高的概率脱离膜表面进入溶液中.利用单分子荧光成像对α-syn解离的观察则发现,随着溶液中的α-syn浓度升高,脂质体上的α-syn解离速率加快.因此高浓度下,α-syn在膜上各区域垂直位置变化促进了蛋白从膜上的解离.结合LipoFRET的实验结果可以推断,α-syn的解离可能是由于不同的α-syn分子膜作用位点互相竞争而导致的解离.这样的特征,可能是体内环境中影响α-syn控制其聚集的重要性质.

α-synuclein(α-syn)is a key protein involved in Parkinson’s disease.There have been many researches about a-syn in recent years.It was suggested that the aggregation of α-syn may induce the lipid membranes to disrupted,which is related to the pathology of neurodegenerative diseases.Thus the studying of the dynamics of α-syn on membranes,especially in the presence of high-concentration protein,is important for understanding its function and its role in the pathology.In this study,we use LipoFRET,a single molecule method based on the principle of energy transfer between the donor labeled on the biomolecule and the quenchers encapsulated in the liposome.The quenchers encapsulated in liposomes attenuate the fluorescence attached to membrane proteins near the membrane,or penetrating in the membranes.If interesting site of membrane protein can be labeled,the LipoFRET could probe positional changes of a single membrane protein in the direction normal to the membrane.In the research of a-syn by LipoFRET,some interesting results can be obtained with different concentrations of protein.On the one hand,with the increase of concentration of α-syn in solution,the centre domain of α-syn can leave the surface of the lipid bilayer and enter into the aqueous solution.However,this domain of α-syn is located around the membrane surface at low concentration.On the other hand,the Nterminus of α-syn with three main positions at low concentration of protein,maintains three but different positions in the membrane at high concentration,where each position is closer to or above the outer surface of liposome.The above phenomena suggeste that the interaction between α-syn and membranes might be weakened with the increase of concentration of protein.At the same time,with single molecule fluorescence imaging,we also observe the promoted dissociation rates for individual fluorophore labeled α-syn from liposomes with high concentration of unlabeled proteins in solution.The result is consistent with the result of our singlemolecule experiment with LipoFRET.Along with the results from LipoFRET,it could be indicated that there is a competition process where each α-syn could be occupied by the other one at high protein concentration,which leads to the dissociation.The concentration-dependent dissociation may be the property that regulates the aggregation of α-syn in vivo,which is one of the important factors that influence the pathology of the neurodegenerative diseases.