Native protein sequences are designed to destabilize folding intermediates
Native protein sequences are designed to destabilize folding intermediates
复制标题
DOI：
10.1021/bi0523714
复制
发表时间：
2006-02-28
期刊：
影响因子：
2.9
通讯作者：
Isogai, Y
中科院分区：
文献类型：
--
作者：
Isogai, Y
构建了抹香鲸脱辅基肌红蛋白的疏水核心突变体，以研究决定其折叠特性的氨基酸序列特征。用Leu替换所有的Ile残基以及用Leu替换所有的Ile和瓦尔残基降低了折叠状态相对于未折叠状态的热力学稳定性，但增加了折叠中间体相对于未折叠状态的稳定性，表明蛋白质核心的氨基酸组成对于蛋白质稳定性和折叠协同性是重要的。为了研究这些疏水残基排列的效果，进一步构建了突变蛋白：野生型肌红蛋白的18个Leu、9个Ile和8个瓦尔残基中的12个位点彼此随机替换，使得氨基酸组成与野生型蛋白相似。在不选择蛋白质性质的情况下获得了四种突变蛋白。与野生型蛋白质相比，这些残基置换类似地导致中间状态和折叠状态相对于未折叠状态的稳定。因此，选择天然氨基酸序列中疏水残基的排列以使折叠中间体不稳定，而不是稳定折叠状态。本研究结果表明，蛋白质折叠的两态转换或不稳定中间体的瞬时形成，这似乎是有效生产功能蛋白质所必需的，一直是天然球状蛋白质分子进化的主要驱动力。
Hydrophobic core mutants of sperm whale apomyoglobin were constructed to investigate the amino acid sequence features that determine the folding properties. Replacements of all of the Ile residues with Leu and of all of the Ile and Val residues with Leu decreased the thermodynamic stability of the folded states against the unfolded states but increased the stability of the folding intermediates against the unfolded states, indicating that the amino acid composition of the protein core is important for the protein stability and folding cooperativity. To examine the effect of the arrangement of these hydrophobic residues, mutant proteins were further constructed: 12 sites out of the 18 Leu, 9 Ile, and 8 Val residues of the wild-type myoglobin were randomly replaced with each other so that the amino acid compositions were similar to that of the wild-type protein. Four mutant proteins were obtained without selection of the protein properties. These residue replacements similarly resulted in the stabilization of both the intermediate and folded states against the unfolded states, as compared to the wild-type protein. Thus, the arrangements of the hydrophobic residues in the native amino acid sequence are selected to destabilize the folding intermediate rather than to stabilize the folded state. The present results suggest that the two-state transition of protein folding or the transient formation of the unstable intermediate, which seems to be required for effective production of the functional proteins, has been a major driving force in the molecular evolution of natural globular proteins.