A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures
A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures
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DOI：
10.1073/pnas.0611503104
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发表时间：
2007-02-20
影响因子：
11.1
通讯作者：
Deniz, Ashok A.
中科院分区：
文献类型：
--
作者：
Mukhopadhyay, Samrat;Krishnan, Rajaraman;Deniz, Ashok A.
酵母朊病毒蛋白Sup 35是一种翻译终止因子，其活性通过螯合成自我永存的淀粉样蛋白来调节。朊病毒决定结构域NM由两个不同的区域组成：淀粉样蛋白N末端结构域（N）和带电增溶中间区域（M）。为了深入了解朊病毒转换，我们使用单分子荧光共振能量转移（SM-FRET）和荧光相关光谱研究单体NM的结构和动力学。在这些实验中，低蛋白浓度防止了专性通路上寡聚体的形成，使我们能够研究与高阶物种分离的早期折叠中间体。SM-FRET实验的双重标记的淀粉样蛋白的核心变体（N21 C/S121 C，保留野生型朊病毒的行为）表明，NM的N区域采用类似于“爆发阶段”的中间体折叠过程中形成的许多球状蛋白，即使它缺乏一个典型的疏水性核心的折叠形式。第21位和第121位残基之间的平均距离约为43 A。这增加了变性剂在一个非合作的方式接近63 A，这表明大量的相互转化的物种，而不是一个小数目的离散单体构象。单标记NM的荧光相关光谱分析显示快速的构象波动在20- 300-ns的时间尺度上。从近端和远端酪氨酸淬灭导致不同的快速和缓慢的波动。我们的研究结果表明，本机单体NM是由一个合奏的结构，有一个崩溃和快速波动的N区并列与一个更广泛的M区。这种集合体的稳定性可能在朊病毒转化中起关键作用。
The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to "burst-phase" intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was approximate to 43 A. This increased with denaturant in a noncooperative fashion to approximate to 63 A, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. Fluorescence correlation spectroscopy analysis of singly labeled NM revealed fast conformational fluctuations on the 20- to 300-ns time scale. Quenching from proximal and distal tyrosines resulted in distinct fast and slower fluctuations. Our results indicate that native monomeric NM is composed of an ensemble of structures, having a collapsed and rapidly fluctuating N region juxtaposed with a more extended M region. The stability of such ensembles is likely to play a key role in prion conversion.