Reshaping the folding energy landscape of human carbonic anhydrase II by a single point genetic mutation Pro237His
Reshaping the folding energy landscape of human carbonic anhydrase II by a single point genetic mutation Pro237His
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通过单点基因突变Pro237His重塑人类碳酸酐酶II的折叠能量景观
DOI：
10.1016/j.biocel.2007.10.022
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发表时间：
2008-01-01
影响因子：
4
通讯作者：
Zhou, Hai-Meng
中科院分区：
文献类型：
--
作者：
Jiang, Yan;Su, Jing-Tan;Zhou, Hai-Meng
人类碳酸酐酶（HCA）II参与多种重要的生物学过程，其基因突变与人类疾病密切相关。在这项研究中，我们研究了基因单点突变P237，这是位于分子的表面，不参与HCA II催化，对HCA II的活性，稳定性和折叠的影响。光谱研究表明，突变导致更多的掩埋色氨酸残基变得可访问的溶剂，并导致NMR信号变得不那么分散，但不影响二级结构或疏水暴露的蛋白质。突变体是不太稳定的野生型酶对节拍和盐酸钆诱导的失活，但其pH适应是类似的野生型。该突变稍微降低了熔融球状中间体的稳定性，但逐渐影响了天然状态的稳定性，从天然状态过渡到中间体的吉布斯自由能减少了10倍。这可能导致易于聚集的熔融球状中间体的积累，其在重折叠期间进一步将蛋白质捕获到非途径聚集体中，并降低体内活性酶的水平。本文的结果表明，P237周围的长环的正确定位可能对HCA II的折叠，特别是活性位点的形成至关重要。(C)2007爱思唯尔有限公司保留所有权利。
Human carbonic anhydrase (HCA) II participates in a variety of important biological processes, and it has long been known that genetic mutations of HCA II are closely correlated to human disease. In this research, we investigated the effects of a genetic single point mutation P237, which is located on the surface of the molecule and does not participate in the HCA II catalysis, on HCA II activity, stability and folding. Spectroscopic studies revealed that the mutation caused more buried Trp residues to become accessible by solvent and caused the NMR signals to become less dispersed, but did not affect the secondary structure or the hydrophobic exposure of the protein. The mutant was less stable than the wild type enzyme against beat- and GdnHCl-induced inactivation, but its pH adaptation was similar to the wild type. The mutation slightly decreased the stability of the molten globular intermediate, but gradually affected the stability of the native state by a 10-fold reduction of the Gibbs free energy for the transition from the native state to the intermediate. This might have led to an accumulation of the aggregation-prone molten globular intermediate, which further trapped the proteins into the off-pathway aggregates during refolding and reduced the levels of active enzyme in vivo. The results herein suggested that the correct positioning of the long loop around P237 might be crucial to the folding of HCA II, particularly the formation of the active site. (C) 2007 Elsevier Ltd. All rights reserved.