A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins.
A combinatorial NMR and EPR approach for evaluating the structural ensemble of partially folded proteins.
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DOI：
10.1021/ja100646t
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发表时间：
2010-06-30
影响因子：
15
通讯作者：
Ulmer, Tobias S.
中科院分区：
文献类型：
--
作者：
Rao, Jampani Nageswara;Jao, Christine C.;Hegde, Balachandra G.;Langen, Ralf;Ulmer, Tobias S.
部分折叠蛋白质是生理性蛋白质折叠和病理性蛋白质错误折叠的重要中间产物，具有疏松或无三级接触的二级结构特征。为了帮助表征这类蛋白质的结构状态(S)，提出了一种新的结构计算方案，该方案结合了脉冲EPR和核磁共振谱的结构约束。建立了α-突触核蛋白(αS)的方法学，当它与洗涤剂月桂酰肌氨酸钠(SLAS)胶束结合时，显示出部分折叠的蛋白质特征。通过将18个EPR得到的电子间自旋标记距离分布与基于核磁共振的二级结构定义和键向量约束相结合，关联了电子间距离，并计算了一组理论系综碱基布居。随后，通过反向计算关联距离分布，得到了一个表示受SLA约束的αS的部分折叠状态的最小基本结构集。由此揭示了一种定义明确的蛋白质-胶束相互作用的惊人变化，其中胶束被两个不同排列的反平行αS螺旋吞噬。该方法进一步提供了主要系综结构状态之间的布居比，而可获得的结构分辨率的限制来自于自旋标记灵活性和二级结构定义中的残余不确定性。为了促进对蛋白质-胶束相互作用的理解，本研究得出结论，与二级结构稳定性明显相反，与二级结构稳定性相反，SLA结合的αS的螺旋动力学与蛋白质诱导的偏离自由胶束尺寸的程度有关。
Partially folded proteins, characterized as exhibiting secondary structure elements with loose or absent tertiary contacts, represent important intermediates in both physiological protein folding and pathological protein misfolding. To aid in the characterization of the structural state(s) of such proteins, a novel structure calculation scheme is presented that combines structural restraints derived from pulsed EPR and NMR spectroscopy. The methodology is established for the protein α-synuclein (αS), which exhibits characteristics of a partially folded protein when bound to a micelle of the detergent sodium lauroyl sarcosinate (SLAS). By combining 18 EPR-derived interelectron spin label distance distributions with NMR-based secondary structure definitions and bond vector restraints, interelectron distances were correlated and a set of theoretical ensemble basis populations was calculated. A minimal set of basis structures, representing the partially folded state of SLAS-bound αS, was subsequently derived by back-calculating correlated distance distributions. A surprising variety of well-defined protein-micelle interactions was thus revealed in which the micelle is engulfed by two differently arranged anti-parallel αS helices. The methodology further provided the population ratios between dominant ensemble structural states, whereas limitation in obtainable structural resolution arose from spin label flexibility and residual uncertainties in secondary structure definitions. To advance the understanding of protein-micelle interactions, the present study concludes by showing that, in marked contrast to secondary structure stability, helix dynamics of SLAS-bound αS correlate with the degree of protein-induced departures from free micelle dimensions.
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