Crystallization and structural analysis of bullfrog red cell L-subunit ferritins.
Crystallization and structural analysis of bullfrog red cell L-subunit ferritins.
复制标题
牛蛙红细胞 L 亚基铁蛋白的结晶和结构分析。
DOI：
10.1002/prot.340180204
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Allewell,NM
中科院分区：
文献类型：
--
作者：
Trikha,J;Waldo,GS;Lewandowski,FA;Ha,Y;Theil,EC;Weber,PC;Allewell,NM
铁蛋白是一种24个亚基的蛋白质，控制着动物、细菌和植物中铁的生物矿化。不同的铁蛋白家族成员的矿化速率不同，特别是在动物铁蛋白的L和H型亚基之间，这两种亚基在不同的细胞类型中表达不同。为了从高度分化的细胞类型中检测铁蛋白，并阐明铁蛋白结构与功能之间的关系，我们克隆了牛蛙红细胞L铁蛋白，并在大肠杆菌中进行了高效表达，并在两种条件下进行了结晶。当MnCl2的浓度与蛋白质的浓度相当，而MgCl2的浓度远高于蛋白质的浓度时，可获得高离子强度的晶体。在两种晶化条件下，晶体均为F432空间群的四方双锥体，晶胞尺寸a=b=c=182±0.5。在锰和硫酸铵存在下得到的晶体衍射率为1.9？，而在镁和酒石酸钠存在时得到的晶体衍射率为1.6？在相似的条件下，也获得了同构晶体，其中Glu−57、−58、−59和−61都被Ala取代。以人肝H亚基的钙盐(Lawson等，Natural(London)349：541-544,1991)为模型，用分子置换的方法解决了野生型含镁L亚基的结构问题。6-2.2ä壳层的结晶R因子为0.21。人类H铁蛋白和牛蛙L铁蛋白的整体折叠相似，主干原子位置的均方根差异为0.97A。最大的结构差异出现在D螺旋和连接四个螺旋束的D和E螺旋的环上。由于红细胞L铁蛋白和肝H铁蛋白在矿化速率和三维结构上都表现出不同，对这些结构进行更详细的比较可能会为构象和功能之间的关系提供新的线索。
Ferritin is a 24 subunit protein that controls biomineralization of iron in animals, bacteria, and plants. Rates of mineralization vary among members of the ferritin family, particularly between L and H type subunits of animal ferritins which are differentially expressed in various cell types. To examine ferritin from a highly differentiated cell type and to clarify the relationship between ferritin structure and function, bullfrog red cell L ferritin has been cloned, overexpressed in E. coli, and crystallized under two conditions. Crystals were obtained at high ionic strength in the presence of MnCl 2 at a concentration comparable to that of the protein and in the presence of MgCl 2 at a concentration much higher than that of the protein. Under both crystallization conditions, the crystals are tetragonal bipyramids in the space group F432 with unit cell dimensions a= b= c= 182±0.5 Å. Crystals obtained in the presence of manganese and ammonium sulfate diffract to 1.9 Å, while those obtained in the presence of magnesium and sodium tartrate diffract to 1.6 Å. Isomorphous crystals have been obtained under similar conditions for a site-directed mutant with a reduced mineralization rate in which Glu− 57,− 58,− 59, and− 61 are all replaced by Ala. The structure of wild type L-subunit with magnesium has been solved by molecular replacement using the calcium salt of human liver H subunit (Lawson et al., Nature (London) 349: 541–544, 1991) as the model. The crystallographic R factor for the 6–2.2 Å shell is 0.21. The overall fold of human H and bullfrog L ferritins is similar with an rms difference in backbone atomic positions of 0.97 Å. The largest structural differences occur in the D helix and the loop connecting the D and E helices of the four helix bundle. Because red cell L ferritin and liver H ferritin show differences in both rates of mineralization and three-dimensional structure, more detailed comparisons of these structures are likely to shed new light on the relationship between conformation and function.© 1994 John Wiley & Sons, Inc.