The extremely slow-exchanging core and acid-denatured state of green fluorescent protein.

Hfsp Journal Pub Date : 2008-12-01 Epub Date: 2008-09-15 DOI:10.2976/1.2976660
Jie-Rong Huang, Shang-Te Danny Hsu, John Christodoulou, Sophie E Jackson
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引用次数: 20

Abstract

Green fluorescent protein (GFP) is a large protein with a complex eleven-stranded beta-barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of HD exchange measurement and nuclear magnetic resonance (NMR) techniques to probe the energy landscape for folding of GFP in further detail. HD exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. HD exchange experiments at high pH establish that there is an extremely slow-exchanging superstable core of amide protons in GFP that are clustered and located in beta-strands 1, 2, 4, 5, and 6. These residues form part of a mini-beta-sheet which we propose constitutes a folding nucleus. Using a pulsed-labeling strategy, the acid-denatured state has been investigated and the residual structure observed in earlier studies shown to locate to beta-strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain.

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绿色荧光蛋白的极慢交换核和酸变性状态。
绿色荧光蛋白(GFP)是一种具有复杂的11链β -桶状结构的大蛋白。先前的研究表明,它具有复杂的折叠能量格局,其中存在多个中间态和一个具有显著残余结构的变性态。在这里,我们使用两种不同类型的HD交换测量和核磁共振(NMR)技术来进一步详细探索绿色荧光蛋白折叠的能量格局。HD交换实验在各种条件下进行,包括不同浓度的变性剂。结果表明,渗透模型主导了交换机制,与已知的GFP稳定性和缓慢展开动力学一致。高pH下的HD交换实验表明,GFP中存在一个极慢交换的超稳定酰胺质子核心,这些质子聚集在β链1、2、4、5和6上。这些残基构成了我们认为构成折叠核的小薄片的一部分。使用脉冲标记策略,研究了酸变性状态,并且在早期研究中观察到的残余结构显示定位于β链1和3。有一些证据表明,这种残余结构是由多肽链的局部疏水崩溃稳定的。
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Hfsp Journal
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