Structure and membrane binding of alpha-synuclein

在多巴胺能神经元中，α-突触核蛋白（aS）在无序的胞质状态和脂质结合状态之间分配。aS与膜磷脂的结合与其突触调节的功能作用有关，但也影响与帕金森病相关的原纤维形成。 Selkoe等人在2011年的一项研究中报道，如果在哺乳动物细胞中表达aS并在没有热变性步骤的情况下纯化，则其采用稳定的四聚体螺旋结构。 我们开发了这种表达系统，但无法复制他们的发现。 然而，我们发现，通过高分辨率NMR光谱和圆二色性（CD）测量，在哺乳动物细胞中发生的N-末端乙酰化影响蛋白质的结构和动力学在自由溶液中，也影响蛋白质的膜结合特性。虽然没有四聚体形式的乙酰化的aS可以被分离，N-末端乙酰化导致的化学位移扰动的前12个残基的蛋白质，逐渐减少与N-末端的距离。 化学位移的变化和骨架3 JHH耦合的小变化的方向是一致的α-螺旋度的前六个残基的aS的增加，虽然仍然存在高度的动态构象紊乱和螺旋结构的采样小于20%。完整蛋白质的化学位移和3 JHH数据实际上与相应的N-末端乙酰化和非乙酰化15-残基合成肽记录的数据无法区分。乙酰化肽上的CD数据和指示α-螺旋特征的弱中等范围NOE接触支持在aS的N-末端的α-螺旋性增加。蛋白质的其余部分具有非常接近无规卷曲值的化学位移值，并且在蛋白质的两种形式之间无法区分。 在乙酰化和非乙酰化的aS之间没有观察到纤颤动力学的显著差异。然而，脂质结合特性的aS强烈影响乙酰化，并表现出独特的行为的前12个残基，指示的起始作用的N-末端残基在“起始-延伸”的过程中结合到膜。 已经开发了一种用于探测α-突触核蛋白的膜结合的新方法，该方法依赖于当小单层囊泡（SUV）中的一小部分脂质含有过氧化烷烃链时其Met残基的自发氧化。 探测突触核蛋白中不同Met残基的氧化速率揭示了N-末端50个残基的蛋白质与膜表面的结合具有很强的协同性。

In dopaminergic neurons, a-synuclein (aS) partitions between a disordered cytosolic state and a lipid-bound state. Binding of aS to membrane phospholipids is implicated in its functional role of synaptic regulation, but also impacts fibril formation associated with Parkinson's disease. A 2011 study by Selkoe et al reported that if aS is expressed in mammalian cells and purified without a heat denaturation step, it adopts a stable tetrameric helical structure. We developed this expression system but were unable to duplicate their findings. However, we found by high-resolution NMR spectroscopy and circular dichroism (CD) measurements, that the N-terminal acetylation which occurs in mammalian cells impacts the protein's structure and dynamics in free solution and also affects the protein's membrane binding properties. While no tetrameric form of acetylated aS could be isolated, N-terminal acetylation resulted in chemical shift perturbations of the first 12 residues of the protein which progressively decreased with distance from the N-terminus. The directions of the chemical shift changes and small changes in backbone 3JHH couplings are consistent with an increase in alpha-helicity of the first six residues of aS, although a high degree of dynamic conformational disorder remains and the helical structure is sampled less than 20%. Chemical shift and 3JHH data for the intact protein are virtually indistinguishable from those recorded for the corresponding N-terminally acetylated and non-acetylated 15-residue synthetic peptides. An increase in alpha-helicity at the N-terminus of aS is supported by CD data on the acetylated peptide, and by weak medium-range NOE contacts indicative of alpha-helical character. The remainder of the protein has chemical shift values that are very close to random coil values and indistinguishable between the two forms of the protein. No significant difference in the fibrillation kinetics were observed between acetylated and non-acetylated aS. However, the lipid binding properties of aS are strongly impacted by acetylation, and exhibit distinct behavior for the first 12 residues, indicative of an initiation role for the N-terminal residues in an "initiation-elongation" process of binding to the membrane. A novel method for probing the membrane binding of alpha-synuclein has been developed which relies on the spontaneous oxidation of its Met residues when a small fraction of the lipids in small unilamellar vesicles (SUVs) contain peroxidized alkane chains. Probing of the rates of oxidation of different Met residues in synuclein revealed a strong degree of cooperativity in the binding of the N-terminal 50 residues of the protein to the membrane surface.