Structure and membrane binding of alpha-synuclein

α-突触核蛋白的结构和膜结合

• 批准号: 8741381
• 负责人: Ad Bax
• 金额: $ 67.68万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8741381
• 关键词: Acetylation; Adopted; Affect; Alkanes; Behavior; Binding; Biological Process; Chemicals; Circular Dichroism; Data; Disease; Exhibits; Heating; Kinetics; Lipid Binding; Lipids; Mammalian Cell; Measurement; Membrane; Membrane Proteins; Methods; N-terminal; NMR Spectroscopy; Parkinson Disease; Peptides; Phospholipids; Process; Property; Protein Dynamics; Proteins; Regulation; Reporting; Resolution; Role; Sampling; Solutions; Structure; Surface; Synapses; System; Vertebral column; alpha synuclein; dopaminergic neuron; insight; meetings; mimetics; mutant; novel; oxidation; protein structure; synthetic peptide; synuclein; unilamellar vesicle

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.

在多巴胺能神经元中，胞质核蛋白（AS）分区之间的胞质状态和脂质结合状态之间的分区。 AS与膜磷脂的结合与其突触调节的功能作用有关，但也影响与帕金森氏病有关的原纤维形成。 Selkoe等人2011年的一项研究报告说，如果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螺旋结构。 我们开发了这个表达系统，但无法复制他们的发现。 然而，我们通过高分辨率的NMR光谱和圆形二色性（CD）测量发现，发生在哺乳动物细胞中的N末端乙酰化会影响蛋白质的结构和自由溶液中的动力学，并影响蛋白质的膜结合特性。虽然没有可以分离的乙酰化的四聚体形式，但N末端乙酰化导致蛋白质的前12个残基的化学位移扰动，该残基随着距离N末端的距离而逐渐减少。 尽管高度高度的动态构象障碍仍然存在，并且螺旋结构的采样小于20％，但化学位移变化的方向和骨干3JHH耦合的小变化与AS的前六个残基的α-壳性增加一致。完整蛋白的化学位移和3JHH数据几乎与相应的N末端乙酰化和非乙酰化15二氧化合成肽记录的数据几乎没有区别。乙酰化肽的CD数据支持N端的N端的α-螺旋，以及指示α-螺旋特征的弱中端NOE接触。其余的蛋白质具有非常接近随机线圈值的化学位移值，并且在两种形式的蛋白质之间都无法区分。 乙酰化和未乙酰化As之间没有观察到纤颤动力学的显着差异。然而，AS的脂质结合特性受乙酰化的强烈影响，并表现出前12个残基的独特行为，这表明N末端残基在与膜结合的“启动 - 延长”过程中具有起始作用。 已经开发了一种探测α-突触核蛋白的膜结合的新方法，该方法依赖于其MET残基的自发氧化，当时一小部分脂质在小的单层囊泡中（SUV）中含有过氧化的烷烃链。 探测不同MET残基在突触核蛋白中的氧化速率表明，蛋白质的N末端50残基与膜表面的N末端50残基的结合具有很强的合作性。