Improving protein crystal quality by selective removal of a Ca2+-dependent membrane-insertion loop
Improving protein crystal quality by selective removal of a Ca2+-dependent membrane-insertion loop
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DOI：
10.1107/s1744309107050993
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发表时间：
2007-11-01
影响因子：
0.9
通讯作者：
Newcomer, Marcia E.
中科院分区：
文献类型：
--
作者：
Neau, David B.;Gilbert, Nathaniel C.;Newcomer, Marcia E.
脂氧合酶（LOX）催化多不饱和膜包埋脂肪酸的区域特异性和立体特异性双加氧。钙依赖性的膜结合功能定位于软珊瑚Plexaura homomalla的8 R-LOX的氨基端C2-like结构域。8 R-LOX的3.2埃晶体结构和光谱数据表明，Ca 2+稳定了两个膜插入环。对晶格中蛋白质包装接触的分析表明，两个环之一的构象使提高X射线数据分辨率的努力变得复杂。对8 R-LOX的缺失突变体进行工程改造，其中相应的膜插入环缺失（Delta 41-45：GSLOX）。膜插入环的去除显著增加了细菌培养物的蛋白质产量和所获得的晶体的质量，导致衍射数据的分辨率改善优于1埃。
Lipoxygenases (LOXs) catalyze the regiospecific and stereospecific dioxygenation of polyunsaturated membrane-embedded fatty acids. A Ca2+-dependent membrane-binding function was localized to the amino-terminal C2-like domain of 8R-lipoxygenase (8R-LOX) from the soft coral Plexaura homomalla. The 3.2 angstrom crystal structure of 8R-LOX and spectroscopic data suggested that Ca2+ stabilizes two membrane-insertion loops. Analysis of the protein packing contacts in the crystal lattice indicated that the conformation of one of the two loops complicated efforts to improve the resolution of the X-ray data. A deletion mutant of 8R-LOX in which the corresponding membrane-insertion loop is absent (Delta 41-45: GSLOX) was engineered. Removal of the membrane-insertion loop dramatically increases the protein yield from bacterial cultures and the quality of the crystals obtained, resulting in a better than 1 angstrom improvement in the resolution of the diffraction data.