Structure and membrane binding of alpha-synuclein

在多巴胺能神经元中，a-突触核蛋白（aS）在无序的胞浆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。 使用一组不同的NMR参数，包括骨干化学位移，homo-和heteropoly J耦合，以及短程NOE，我们已经开发了一个详细的描述的骨干扭转角分布在残基特定的水平，这种内在无序的蛋白质。 虽然从经典的螺旋库的偏差是小的，它们是统计上相当显着的，并提供了第一个详细的视图的骨干扭转角采样的本质上无序的蛋白质。 这些骨干扭转角分布交叉验证大大优于合奏模型描述的全链，在其他几个实验室开发的方法。

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. Using a diverse set of NMR parameters, including backbone chemical shifts, homo- and heteronuclear J couplings, as well as short range NOEs, we have developed a detailed description of the backbone torsion angle distribution at the residue-specific level for this intrinsically disordered protein. Although the deviations from classical coil libraries are small, they are statistically quite significant and provide a first detailed view of the backbone torsion angles sampled by an intrinsically disordered protein. These backbone torsion angle distributions cross validate considerably better than ensemble model descriptions of the full chain, an approach developed in several other laboratories.