Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed α/β protein, Escherichia coli thioredoxin
Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed α/β protein, Escherichia coli thioredoxin
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DOI：
10.1021/bi9805083
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发表时间：
1998-07-14
期刊：
影响因子：
2.9
通讯作者：
Chaffotte, AF
中科院分区：
文献类型：
--
作者：
Georgescu, RE;Li, JH;Chaffotte, AF
氧化型大肠杆菌硫氧还蛋白（Oxidized Escherichia coli thioredoxin，Trx）是一种由108个氨基酸残基组成的小分子蛋白质（C32-C35基本上参与活性）和没有辅基部分，辅基部分折叠成包含在许多较大蛋白质中发现的侧接螺旋的中心扭曲β-折叠的结构基序，采用新开发的活性位点滴定法和连续或停流（SF）法结合圆二色谱（CD）研究了Trx的体外复性动力学和荧光（F1）光谱。这些研究揭示了早期折叠中间体的存在与“熔融球或预熔融球”的特点。在222 nm处的椭圆率的测量表明，约68%的总变化与复性发生在死时间（4毫秒）的停流仪器，表明大量的二级结构的形成。使用组合的连续和停流方法重建爆发中间体的远紫外CD光谱显示形成了定义的二级结构，其包含比天然状态更多的β结构，使用SF远紫外CD和FI在宽范围内进行动力学测量（0.087-6 M）两种温度下的GuHCl浓度（6 - 20 ℃）证明了在4 ms死时间期间形成的群体包含主要通过疏水相互作用稳定的多种物质，沿着交替的路径进一步折叠。这些物种之一导致直接和快速的天然状态，如活性位点滴定所示，而其他两个折叠成第四个中间体，缓慢转化为天然蛋白质。双跳实验表明，折叠行为的异质性的结果从脯氨酸异构化发生在未折叠状态。相反，爆发中间体的积累不依赖于脯氨酸异构化。
Oxidized Escherichia coli thioredoxin (Trx) is a small protein of 108 residues with one disulfide bond (C32-C35 essentially involved in the activity) and no prosthetic moieties, which folds into a structural motif containing a central twisted beta-sheet flanked by helices that is found in many larger proteins, The kinetics of refolding of Trx in vitro have been investigated using a newly developed active site titration assay and continuous or stopped-flow (SF) methods in conjunction with circular dichroism (CD) and fluorescence (F1) spectroscopy. These studies revealed the presence of early folding intermediates with "molten globule or pre-molten globule" characteristics. Measurements of the ellipticity at 222 nm indicated that about 68% of the total change associated with refolding occurred during the dead time (4 ms) of the stopped-flow instrument, suggesting the formation of substantial secondary structure. The reconstruction of the far-UV CD spectrum of the burst intermediate using combined continuous and stopped-flow methods showed the formation of a defined secondary structure that contains more beta-structure than the native state, Kinetic measurements using SF far-UV CD and Fl over a wide range (0.087-6 M) of GuHCl concentrations at two temperatures (6 and 20 degrees C) demonstrated that the population formed during the 4 ms dead time contained multiple species that are stabilized mainly by hydrophobic interactions and undergo further folding along alternative pathways. One of these species leads directly and rapidly to the native state as demonstrated by active site titration, while the two others fold into a fourth intermediate that is slowly converted to the native protein. Double-jump experiments suggest that the heterogeneity in folding behavior results from proline isomerizations occurring in the unfolded state. Conversely, the accumulation of the burst intermediate does not depend on proline isomerizations.