Design of a Novel Heme Protein with a Non-Heme Globin Scaffold
Design of a Novel Heme Protein with a Non-Heme Globin Scaffold
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DOI：
10.1021/bi900518q
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发表时间：
2009-09-01
期刊：
影响因子：
2.9
通讯作者：
Ishida, Manabu
中科院分区：
文献类型：
--
作者：
Isogai, Yasuhiro;Ishida, Manabu
铁原卟啉IX（血红素）的结合位点的设计和嵌入在光合作用的非血红素蛋白，藻蓝蛋白，形成一个球蛋白样的骨架结构，称为珠蛋白折叠，但缺乏序列相似性的珠蛋白家族含有肌红蛋白和血红蛋白。基于藻蓝蛋白和肌红蛋白分子的结构比对，近端和远端His残基在藻蓝蛋白序列中重新定位用于血红素连接。通过在藻蓝蛋白核心中的几个残基替换，在His残基周围产生血红素结合口袋。合成的藻蓝蛋白变体，命名为HPY，每个蛋白分子结合一个血红素，并显示六配位血红素蛋白的光谱特征。血红素结合的HPY表现出氧化还原活性，其对标准氢电极的电化学中点电位为-130 mV，这比天然肌红蛋白的电位低约200 mV，但比设计的具有四螺旋束或珠蛋白支架的血红素蛋白的典型值高50 mV。HPY也显示出类似天然的折叠特性，与这些设计的血红素蛋白相反。然而，与天然球蛋白不同，HPY结合的亚铁血红素很快被空气再氧化，不能稳定地结合O（2）。目前的结果表明，一个非珠蛋白的珠蛋白折叠是适合于结合血红素，但不足以为可逆的O（2）结合和肌红蛋白的功能。通过与天然血红蛋白的比较，可能会对天然血红素蛋白功能的本质特征产生新的认识。
A binding site for iron protoporphyrin IX (heme) was designed and embedded in a photosynthetic non-heme protein, phycocyanin, which forms a globin-like backbone structure, called a globin fold, but lacks sequence similarity to the globin family containing myoglobins and hemoglobins. Based oil the structural alignment of the phycocyanin and myoglobin molecules, the proximal and distal His residues were repositioned in the phycocyanin sequence for heme ligation. The heme-binding pocket was created around the His residues by several residue replacements in the phycocyanin core, The synthesized phycocyanin variant, designated as HPY, bound one heme per protein molecule and showed spectroscopic features characteristic of six-coordinated heme proteins. The heme-binding HPY exhibited redox activity with an electrochemical midpoint potential of -130 mV against the standard hydrogen electrode, which was approximately 200 mV lower than the potential of natural myoglobins but 50 mV higher than the typical values of designed heme proteins with four-helix bundle or globin scaffolds. HPY also displayed native-like folding properties, in contrast to these designed heme proteins. However, the bound ferrous heme of HPY was quickly reoxidized by air and did not stably bind O(2), unlike the natural globins. The present results demonstrated that the globin fold of a non-globin protein is suitable for binding heme but is not sufficient for the reversible O(2) binding and myoglobin functions. The comparison of HPY with the natural globins may yield new insights into the essential features for realizing the natural heme protein functions.