Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.
Conformational equilibria in monomeric alpha-synuclein at the single-molecule level.
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DOI：
10.1371/journal.pbio.0060006
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发表时间：
2008-01
期刊：
影响因子：
9.8
通讯作者：
Samorì B
中科院分区：
文献类型：
--
作者：
Sandal M;Valle F;Tessari I;Mammi S;Bergantino E;Musiani F;Brucale M;Bubacco L;Samorì B
人α-突触核蛋白（αSyn）是一种天然未折叠蛋白，其聚集成淀粉样原纤维参与帕金森病的病理。充分理解导致聚集状态的早期中间体的结构和动力学是试图破译αSyn聚集和原纤维形成的分子机制的研究人员的一个至关重要的问题。迄今为止用于解决这一问题的传统体技术指出αSyn独特的构象性质与其聚集倾向之间存在直接关联，但这些技术只能提供单体和低聚物的整体平均信息。因此，它们不能描述触发聚合过程的构象平衡的全部复杂性。我们应用原子力显微镜为基础的单分子力学展开方法研究了人类αSyn野生型和突变型的构象平衡。在单分子水平上表征了单体αSyn的构象非均质性。三种主要的构象，包括无序和“β样”结构，在没有任何低聚可溶形式干扰的情况下直接观察和量化。“β样”结构的相对丰度在不同条件下显著增加，促进αSyn聚集：Cu2+的存在、致病的A30P突变和高离子强度。这种方法可以在单分子水平上探索蛋白质的完整构象空间，甚至可以检测到人口稀少的构象，并测量它们在各种重要生物学条件下的分布。据我们所知，我们首次提出了构象平衡的证据，该平衡控制着特定类别的单体αSyn构象的种群，与已知的促进聚集体形成的条件正相关。从而提供了一种新的工具来直接测试突变和药理策略对单体αSyn构象平衡的影响。天然非结构化蛋白质违背了蛋白质科学的经典“一个序列-一个结构”范式。在病理条件下，这些蛋白质的单体可以在细胞中聚集，这一过程是阿尔茨海默氏症和帕金森等神经退行性疾病的基础。聚集过程的关键步骤——错误折叠的中间产物的形成——仍然不清楚。为了阐明这一过程，我们通过机械拉伸αSyn蛋白的单分子并记录其力学性质，表征了αSyn蛋白的折叠和构象多样性。αSyn是一种与帕金森病有关的天然非结构蛋白。这些实验使我们能够直接观察和量化在体外生理条件下同时存在于αSyn样品中的三种主要构象。我们发现一类构象，“β样”结构，与αSyn聚集直接相关。事实上，它们的相对丰度在三种已知的促进αSyn原纤维形成的不同条件下急剧增加。我们预计，α - syn具有“β样”结构的临界浓度必须达到触发原纤维形成。因此，这个临界浓度是由化学平衡控制的。新的药理学策略现在可以通过靶向这种平衡，在聚集过程发生之前，在上游发挥作用。为此，单分子力谱可以成为定制和测试新药物的有效工具。单分子研究检测α-突触核蛋白单体平衡中的结构和非结构构象。β样构象随着病理突变和其他已知促进聚集的条件而增加。
Human α-Synuclein (αSyn) is a natively unfolded protein whose aggregation into amyloid fibrils is involved in the pathology of Parkinson disease. A full comprehension of the structure and dynamics of early intermediates leading to the aggregated states is an unsolved problem of essential importance to researchers attempting to decipher the molecular mechanisms of αSyn aggregation and formation of fibrils. Traditional bulk techniques used so far to solve this problem point to a direct correlation between αSyn's unique conformational properties and its propensity to aggregate, but these techniques can only provide ensemble-averaged information for monomers and oligomers alike. They therefore cannot characterize the full complexity of the conformational equilibria that trigger the aggregation process. We applied atomic force microscopy–based single-molecule mechanical unfolding methodology to study the conformational equilibrium of human wild-type and mutant αSyn. The conformational heterogeneity of monomeric αSyn was characterized at the single-molecule level. Three main classes of conformations, including disordered and “β-like” structures, were directly observed and quantified without any interference from oligomeric soluble forms. The relative abundance of the “β-like” structures significantly increased in different conditions promoting the aggregation of αSyn: the presence of Cu2+, the pathogenic A30P mutation, and high ionic strength. This methodology can explore the full conformational space of a protein at the single-molecule level, detecting even poorly populated conformers and measuring their distribution in a variety of biologically important conditions. To the best of our knowledge, we present for the first time evidence of a conformational equilibrium that controls the population of a specific class of monomeric αSyn conformers, positively correlated with conditions known to promote the formation of aggregates. A new tool is thus made available to test directly the influence of mutations and pharmacological strategies on the conformational equilibrium of monomeric αSyn. Natively unstructured proteins defy the classical “one sequence–one structure” paradigm of protein science. In pathological conditions, monomers of these proteins can aggregate in the cell, a process that underlies neurodegenerative diseases such as Alzheimer and Parkinson. A key step in the aggregation process—the formation of misfolded intermediates—remains obscure. To shed light on this process, we characterized the folding and conformational diversity of αSyn, a natively unstructured protein involved in Parkinson disease, by mechanically stretching single molecules of this protein and recording their mechanical properties. These experiments permitted us to observe directly and quantify three main classes of conformations that, under in vitro physiological conditions, exist simultaneously in the αSyn sample. We found that one class of conformations, “β-like” structures, is directly related to αSyn aggregation. In fact, their relative abundance increases drastically in three different conditions known to promote the formation of αSyn fibrils. We expect that a critical concentration of αSyn with a “β-like” structure must be reached to trigger fibril formation. This critical concentration is therefore controlled by a chemical equilibrium. Novel pharmacological strategies can now be tailored to act upstream, before the aggregation process ensues, by targeting this equilibrium. To this end, single-molecule force spectroscopy can be an effective tool to tailor and test new pharmacological agents. A single-molecule study detects structured and unstructured conformers in equilibrium in monomeric α-synuclein. The β-like conformers increase with pathological mutations and under other conditions known to promote aggregation.
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DOI：
10.1073/pnas.0407146102
发表时间：
2005-02-01
影响因子：
11.1
作者：
Bertoncini, CW;Jung, YS;Zweckstetter, M
通讯作者：
Zweckstetter, M
影响因子：
82.9
作者：
Conway, KA;Harper, JD;Lansbury, PT
通讯作者：
Lansbury, PT
影响因子：
4.8
作者：
Bertoncini, CW;Fernandez, CO;Zweckstetter, M
通讯作者：
Zweckstetter, M
影响因子：
2.9
作者：
Conway, KA;Harper, JD;Lansbury, PT
通讯作者：
Lansbury, PT
DOI：
10.1073/pnas.97.2.571
发表时间：
2000-01-18
影响因子：
11.1
作者：
Conway, KA;Lee, SJ;Lansbury, PT
通讯作者：
Lansbury, PT