Stacked sets of parallel, in-register beta-strands of beta2-microglobulin in amyloid fibrils revealed by site-directed spin labeling and chemical labeling.
Stacked sets of parallel, in-register beta-strands of beta2-microglobulin in amyloid fibrils revealed by site-directed spin labeling and chemical labeling.
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DOI：
10.1074/jbc.m110.117234
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发表时间：
2010-05-28
期刊：
影响因子：
--
通讯作者：
Langen R
中科院分区：
文献类型：
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作者：
Ladner CL;Chen M;Smith DP;Platt GW;Radford SE;Langen R
β2-微球蛋白（β 2 m）是一种99个残基的蛋白质，具有免疫球蛋白折叠，在透析相关淀粉样变性中形成富含β-片层的淀粉样纤维。在这里，通过定点自旋标记和使用19个位点特异性半胱氨酸变体用N-乙基马来酰亚胺修饰的可及性，探测了由β 2 m体外形成的具有长直形态的淀粉样蛋白原纤维内侧链的环境和可及性。这些纤维的连续波电子顺磁共振光谱显示，与其他β 2 m聚集体相比，核心主要以平行、对齐排列的方式组织。涉及大部分多肽序列的平行、对齐β链的连续阵列与冷冻电子显微镜结构不一致，冷冻电子显微镜结构揭示了基于亚基重复的结构。为了协调这些数据，确定引起自旋交换所需的紧密接近的自旋的数量。对模型蛋白质系统的系统研究表明，四个自旋标记的并置足以产生交换窄化。结合关于侧链移动性和可及性的信息，我们提出β 2 m的淀粉样纤维由大约六个β 2 m单体组成，这些单体以平行的、对齐的阵列堆叠在一起。结果表明，淀粉样纤维的组织结构比通常提出的手风琴样β-三明治结构更复杂。
β2-microglobulin (β2m) is a 99-residue protein with an immunoglobulin fold that forms β-sheet-rich amyloid fibrils in dialysis-related amyloidosis. Here the environment and accessibility of side chains within amyloid fibrils formed in vitro from β2m with a long straight morphology are probed by site-directed spin labeling and accessibility to modification with N-ethyl maleimide using 19 site-specific cysteine variants. Continuous wave electron paramagnetic resonance spectroscopy of these fibrils reveals a core predominantly organized in a parallel, in-register arrangement, by contrast with other β2m aggregates. A continuous array of parallel, in-register β-strands involving most of the polypeptide sequence is inconsistent with the cryoelectron microscopy structure, which reveals an architecture based on subunit repeats. To reconcile these data, the number of spins in close proximity required to give rise to spin exchange was determined. Systematic studies of a model protein system indicated that juxtaposition of four spin labels is sufficient to generate exchange narrowing. Combined with information about side-chain mobility and accessibility, we propose that the amyloid fibrils of β2m consist of about six β2m monomers organized in stacks with a parallel, in-register array. The results suggest an organization more complex than the accordion-like β-sandwich structure commonly proposed for amyloid fibrils.