Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.
Membrane-bound alpha-synuclein forms an extended helix: long-distance pulsed ESR measurements using vesicles, bicelles, and rodlike micelles.
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DOI：
10.1021/ja804517m
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发表时间：
2008-10-01
影响因子：
15
通讯作者：
Eliezer, David
中科院分区：
文献类型：
--
作者：
Georgieva, Elka R.;Ramlall, Trudy F.;Borbat, Peter P.;Freed, Jack H.;Eliezer, David
我们应用脉冲偶极ESR光谱（ku波段DEER）来阐明帕金森病相关蛋白α -突触核蛋白（αS）与单层磷脂小泡、棒状SDS胶束或脂质小束结合的整体构象。通过测量引入的氮氧化物自旋标签对之间长达~7 nm的距离，我们表明，在所有研究情况下，距离都接近于单个连续螺旋的期望。特别是，我们发现位点24和72之间的距离为7.5 nm；位点24和61之间相距5.5 nm；位点35和50之间2纳米。我们得出的结论是，αS不保留具有两个反平行螺旋的“发夹”结构，这与我们之前的发现一致，即蛋白质的几何形状是由表面拓扑决定的，而不是由螺旋间连接体限制的。虽然在膜结合αS结构中存在局部螺旋不连续的可能性，但我们的数据更符合一个完整的螺旋。重要的是，我们证明了bicelles产生与脂质体非常相似的结果，同时在实验可获得的距离范围和分辨率方面提供了重大改进，因此是用于脉冲偶极ESR光谱的极好的脂膜模拟物。
We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson’s disease-associated protein, alpha-synuclein (αS) bound to small unilamellar phospholipid vesicles, rod-like SDS micelles, or lipid bicelles. By measuring distances as long as ~7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that αS does not retain a ‘hairpin’ structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein’s geometry is determined by the surface topology rather than being constrained by the inter-helix linker. While the possibility of local helix discontinuities in the structure of membrane-bound αS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.
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