Resurrecting abandoned proteins with pure water: CD and NMR studies of protein fragments solubilized in salt-free water
Resurrecting abandoned proteins with pure water: CD and NMR studies of protein fragments solubilized in salt-free water
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DOI：
10.1529/biophysj.106.093187
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发表时间：
2006-12-01
影响因子：
3.4
通讯作者：
Song, Jianxing
中科院分区：
文献类型：
--
作者：
Li, Minfen;Liu, Jingxian;Song, Jianxing
大肠杆菌细胞中表达的许多蛋白质形成在缓冲液中既不可折叠也不可溶的包涵体。非常令人惊讶的是，我们最近发现，我们拥有的所有11种缓冲液不溶性蛋白质片段/结构域，具有细胞功能、位置和分子大小的巨大多样性，可以容易地溶解在无盐水中。圆二色谱（CD）和核磁共振（NMR）表征将这些蛋白质分为三组：第1组，CD不具有二级结构，具有窄分散但尖锐的H-1-N-15杂原子单量子相关（HSQC）峰：第2组，CD具有二级结构，但HSQC峰变宽，因此只有一小组峰可检测到;第3组，具有CD二级结构和分离良好的HSQC峰。有趣的是，我们没有发现任何具有紧密三级包装的蛋白质。因此，我们提出，缓冲液不溶性蛋白质可能缺乏内在的能力，达到或/和保持良好的包装构象，因此被困在部分折叠状态与许多疏水性侧链暴露于散装溶剂。因此，非常低的离子强度足以筛选出固有的排斥相互作用，并因此允许发生疏水性聚簇/聚集。令人惊讶的是，在纯水中，蛋白质似乎有可能通过利用固有的排斥相互作用来抑制有吸引力的疏水性聚集，从而表现出其全谱的结构状态。我们的发现不仅为不溶性蛋白质的性质提供了新的见解，而且还揭示了我们所知道的与蛋白质相关的先前未知的机制。
Many proteins expressed in Escherichia coli cells form inclusion bodies that are neither refoldable nor soluble in buffers. Very surprisingly, we recently discovered that all 11 buffer-insoluble protein fragments/ domains we have, with a great diversity of cellular function, location, and molecular size, could be easily solubilized in salt-free water. The circular dichroism ( CD) and NMR characterization led to classification of these proteins into three groups: group 1, with no secondary structure by CD and with narrowly-dispersed but sharp H-1-N-15 heteronuclear single quantum correlation (HSQC) peaks; group 2, with secondary structure by CD but with HSQC peaks broadened and, consequently, only a small set of peaks detectable; and group 3, with secondary structure by CD and also well-separated HSQC peaks. Intriguingly, we failed to find any protein with a tight tertiary packing. Therefore, we propose that buffer-insoluble proteins may lack intrinsic ability to reach or/and to maintain a well-packed conformation, and thus are trapped in partially-folded states with many hydrophobic side chains exposed to the bulk solvent. As such, a very low ionic strength is sufficient to screen out intrinsic repulsive interactions and, consequently, allow the hydrophobic clustering/aggregation to occur. Marvelously enough, it appears that in pure water, proteins have the potential to manifest their full spectrum of structural states by utilizing intrinsic repulsive interactions to suppress the attractive hydrophobic clustering. Our discovery not only gives a novel insight into the properties of insoluble proteins, but also sheds the first light that we know of on previously unknown regimes associated with proteins.