Protein Misfolding and Aggregation

α-突触核蛋白(α-syn)是一种富含140个残基的蛋白，功能不明确，主要分布于突触前神经元终末。值得注意的是，大脑中聚集或淀粉样α-syn的存在是帕金森病的一个特征，其构象和聚集动力学与膜密切相关。虽然已有大量的研究工作致力于了解蛋白质构象动力学对膜相互作用的影响，但仍有一个核心问题是蛋白质缔合如何影响磷脂双层结构和性质。 1.α-突触核蛋白在脂膜界面的中子反射光谱研究 在以前的工作中，我们已经分别从残基水平和双层角度用定点荧光光谱和中子反射仪(NR)评估了α-SYN的膜渗透深度。虽然从两种不同的膜模型--单层囊泡和稀疏拴系双层类脂膜(TBLM)获得的穿透深度高度一致，但NR表征的双层以上蛋白质的分布有些出乎意料。具体地说，α-SYN既延伸到碳氢化合物核心也延伸到主体溶剂区。通常认为，前100个残基是膜相互作用的，采用a-螺旋构象，而酸性的C-末端尾部在溶液中保持无序。嵌入的蛋白质区域的厚度约为15埃，与预期的阿尔法螺旋相当。然而，观察到的进入主体溶剂的蛋白质密度似乎比最后40个C末端残基的预期要大得多。需要实验证据来确定说明不同密度区域的特定残留物。 为了描述特定蛋白质区域的参与，我们产生了一个节段性同位素标记的α-SYN变异体，其中N-末端(残基1-86)和C-末端(残基87-140)分别氢化和质子化。有了N-末端区域的氚，质子化的脂类和多肽之间的对比将很容易区分。使用质谱仪，我们估计氚的水平至少在90%到96%之间。初始结扎反应快速有效(约30min)。在蛋白质浓度低至10 nM的情况下，已经成功地收集了NR数据。目前正在努力优化样品制备和研究磷脂头基对tBLM上α-SYN结构的影响。 2.α-突触核蛋白的膜重塑及其对淀粉样蛋白形成的影响 一个新的观点是，α-syn可以强烈地影响磷脂双层的结构和性质。最近的例子包括膜变薄、膜曲率产生以及管状结构的形成。阴离子磷脂的存在，如磷脂酰甘油(PG)、磷脂酰丝氨酸(PS)或磷脂酸(PA)的存在，以及α-螺旋结构的折叠被认为是膜结合和α-SYN重塑(变形)所必需的。膜的形状和双层的完整性在细胞活动中是至关重要的，例如细胞内的囊泡运输。因此，有必要假设，α-合成酶弯曲和重塑细胞膜是其生理和病理功能的一部分。 最近的报道发现，α-syn诱导含有阴离子脂类的囊泡中的膜微管形成，尤其是popg(1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1‘-rac-glycerol)).。与带负电荷的脂类的相互作用和α-syn形成的α-螺旋结构被认为是重要的因素。出乎意料的是，我们发现平均直径为100 nm的POPC(1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine)囊泡在α-SYN的作用下迅速(秒)重塑成管状结构，这一点通过负染的透射电子显微镜观察到。即使在低含量的α-syn(数百纳摩尔)存在的情况下，在各种不同的脂蛋白比下，也可以通过电子显微镜清楚地观察到小管。此外，管状抑制α-SYN淀粉样蛋白的形成。这些结果似乎与目前关于膜曲率产生机制的假说相矛盾，该假说涉及膜缔合时形成两亲性螺旋结构，因为在POPC囊泡存在的情况下，α-SYN的二级结构变化不能被圆二色谱检测到。包括荧光光谱在内的其他技术目前正被用来绘制特定的相互作用区域，以揭开这一明显的悖论。

alpha-Synuclein (alpha-syn) is an abundant 140 residue protein of ill-defined function enriched in the presynaptic neuronal terminals. Notably, the presence of aggregated or amyloid alpha-syn in the brain is a hallmark of Parkinson's disease and its conformation and aggregation kinetics are intimately tied to membranes. While substantial research efforts have been geared towards the understanding of protein conformational dynamics upon membrane interaction, a central question that remains is how protein association influences phospholipid bilayer structure and properties. 1. Neutron Reflectometry Studies of alpha-Synuclein at the Lipid Membrane Interface In prior work, we have assessed the membrane penetration depth of alpha-syn on the residue-level and from the perspective of the bilayer by using site-specific fluorescence spectroscopy and neutron reflectometry (NR), respectively. While the penetration depths obtained from two different membrane models, unilamellar vesicles and sparsely tethered bilayer lipid membrane (tBLM), were highly consistent, the profile for protein occupancy above the bilayer characterized by NR were somewhat unexpected. Specifically, alpha-syn extends into the hydrocarbon core as well as into the bulk solvent region. It is generally thought that the first 100 residues are membrane interacting, adopting an -helical conformation whereas the acidic C-terminal tail remaining disordered in solution. The thickness of the embedded protein region is around 15 angstroms comparable to that would be expected of an alpha-helix. However, the observed protein density out into the bulk solvent appears to be more extensive than would be expected for the last 40 C-terminal residues. Experimental evidence is needed to determine the specific residues that account for the different density regions. Towards the aim to delineate the involvement of specific protein regions, we have produced a segmental isotopically-labeled alpha-syn variant where the N-terminal (residues 1-86) and C-terminal regions (residues 87-140) are deuterated and protonated, respectively. Having the N-terminal region deuterated, the contrast between the protonated lipid and the peptide would be easily distinguishable. Using mass spectrometry, we estimate the level of deuteration to be at least 90 to 96%. Initial ligation reaction was rapid and efficient (< 30 min). NR data have been successfully collected using protein concentration as low as 10 nM. Ongoing efforts are geared toward optimization of sample preparation and investigation of effects of phospholipid headgroups on alpha-syn structure on the tBLM. 2. Membrane Remodeling by alpha-Synuclein and Effects on Amyloid Formation An emerging view is that alpha-syn can strongly influence the structure and properties of phospholipid bilayers. Recent examples include membrane thinning, membrane curvature generation, as well as formation of tubular structures. Presence of anionic phospholipids, e.g. phosphatidylglycerol (PG), phosphatidylserine (PS), or phosphatidic acid (PA), and folding of alpha-helical structure are thought to be essential for membrane binding and remodeling (deformation) by alpha-syn. Membrane shapes along with bilayer integrity are crucial in cellular activities such as intracellular vesicular transport. Accordingly, it is compelling to hypothesize that alpha-syn bends and remodels membranes as part of its physiological as well as pathological function. Recent reports have found that alpha-syn induces membrane tubulation in vesicles containing anionic lipids, especially POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol)). Interaction with negatively charged lipids and the formation of alpha-helix structure by alpha-syn are proposed to be important factors. Unexpectedly, we found that POPC (1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine) vesicles of average diameter 100 nm are remodeled rapidly ( seconds) into tube-like structures by alpha-syn as visualized by negative staining transmission electron microscopy (TEM). Even in the presence of low amounts of alpha-syn (hundreds of nanomolar), tubules were clearly observed by TEM under a wide variety of lipid-to-protein ratios. Moreover, tubulation inhibits alpha-syn amyloid formation. These results appear to contradict the current hypothesis for membrane curvature generation mechanism which involves the formation of amphipathic helical structure upon membrane association as secondary structure changes of alpha-syn in the presence of POPC vesicles are undetectable by circular dichroism spectroscopy. Other techniques including fluorescence spectroscopies are currently being employed to map the specific interacting region in order to unravel this apparent paradox.