Structure and membrane binding of alpha-synuclein

在多巴胺能神经元中，α-突触核蛋白(AS)在无序的胞浆状态和脂质结合状态之间进行划分。AS与膜磷脂的结合与其突触调节的功能有关，但也影响与帕金森病相关的纤维形成。 2011年，Selkoe等人的一项研究报告称，如果AS在哺乳动物细胞中表达并在没有热变性步骤的情况下进行纯化，它将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，我们通过高分辨核磁共振光谱和圆二色谱(CD)测量发现，哺乳动物细胞中发生的N-末端乙酰化作用影响了蛋白质在自由溶液中的结构和动力学，也影响了蛋白质的膜结合性质。虽然没有分离到四聚体形式的乙酰化AS，但N-末端的乙酰化导致了蛋白质前12个残基的化学位移扰动，随着距离N-末端的距离逐渐减少。主链3JHH偶联的化学位移变化和微小变化的方向与As的前六个残基的α-螺旋度增加一致，尽管动态构象无序程度仍然很高，螺旋结构的采样低于20%。完整蛋白质的化学位移和3JHH数据与相应的N-末端乙酰化和非乙酰化15个残基的合成肽的数据几乎无法区分。乙酰化肽上的CD数据和表明α-螺旋性质的弱的中程NOE接触支持了AS N-末端的α-螺旋性的增加。蛋白质的其余部分具有非常接近随机卷曲值的化学移动值，并且无法区分蛋白质的两种形式。乙酰化和非乙酰化AS的原纤化动力学没有显著差异。然而，AS的脂质结合性质受到乙酰化的强烈影响，并对前12个残基表现出不同的行为，表明N末端残基在与膜结合的“起始-延伸”过程中起到了启动作用。 使用一组不同的核磁共振参数，包括骨架化学位移、同核和异核J偶合以及短程NOE，我们已经详细描述了这种内在无序蛋白质在残基特定水平上的骨架扭转角分布。尽管与经典线圈文库的偏差很小，但它们在统计学上相当重要，并提供了由内在无序蛋白质采样的骨架扭转角的第一个详细视图。这些主干扭角分布的交叉验证比全链的系综模型描述要好得多，这是其他几个实验室开发的一种方法。

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.