Structural dissection of alkaline-denatured pepsin
Structural dissection of alkaline-denatured pepsin
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DOI：
10.1155/2004/769354
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发表时间：
2004-01-01
影响因子：
--
通讯作者：
Konno, T
中科院分区：
文献类型：
--
作者：
Kamatari, YO;Dobson, CM;Konno, T
胃蛋白酶是一种胃天冬氨酸蛋白酶，是一种酶原来源的蛋白质，在pH值为6-7的条件下发生不可逆碱性变性。详细了解碱变性状态的结构是了解活性酶形成机制的重要一步。许多研究表明，胃蛋白酶的碱变性状态(I-P状态)由一个紧凑的C-末端小叶和一个基本上无结构的N-末端小叶组成。在本研究中，我们更详细地研究了I-P状态下的残基结构，使用有限的蛋白质分解从这种部分变性的胃酶中分离和鉴定了一个紧密折叠的核心区。分离的核心区对应于胃酶分子的141个C-末端残基，在完全天然状态下形成结构的两个叶之一。然而，使用核磁共振和圆二色谱的比较研究表明，N-末端小叶为胃酶的I-P状态贡献了大量的额外残基结构。此外，CD谱还表明，当胃酶的N-末端叶展开或通过蛋白分解去除时，在结构的C-末端叶中存在显著的非天然α-螺旋结构。这项研究表明，I-P状态下的胃蛋白酶结构比完全折叠的C-末端叶连接到非结构化N-末端叶的结构要复杂得多。在这种状态下的“错误折叠”可能会抑制蛋白质在返回到稳定自然状态的条件下正确的重折叠。
Pepsin, a gastric aspartic proteinase, is a zymogen-derived protein that undergoes irreversible alkaline denaturation at pH 6-7. Detailed knowledge of the structure of the alkaline-denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline-denatured state of pepsin (the I-P state) is composed of a compact C-terminal lobe and a largely unstructured N-terminal lobe. In the present study, we have investigated the residual structure in the I-P state in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C-terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N-terminal lobe contributes a substantial amount of additional residual structure to the I-P state of pepsin. CD spectra indicate in addition that significant non-native a-helical structure is present in the C-terminal lobe of the structure when the N-terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the I-P state is significantly more complex than that of a fully folded C-terminal lobe connected to an unstructured N-terminal lobe. The "misfolding" in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.