Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil
Metal-ion-dependent GFP emission in vivo by combining a circularly permutated green fluorescent protein with an engineered metal-ion-binding coiled-coil
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DOI：
10.1021/ja0685102
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发表时间：
2007-09-19
影响因子：
15
通讯作者：
Tanaka, Toshiki
中科院分区：
文献类型：
--
作者：
Mizuno, Toshihisa;Murao, Kaori;Tanaka, Toshiki
金属离子的配位对蛋白质的结构和功能起着重要的作用。本文构建了一种融合蛋白，由一个全新设计的金属离子结合的三聚体卷曲线圈和一个圆形排列的绿色荧光蛋白（cpGFP）组成，其中cpGFP的荧光发射是由金属离子配位诱导的。通过GGSGG连接GFP（uv）的原始N端和c端，并将其切割在Asp（190）和Gly（191）之间，构建了环状排列的GFP(191)cpGFP（190）。金属离子结合的螺旋线圈，IZ-HH，被设计成具有三个α -螺旋结构，在螺旋线圈结构的疏水核心有12个他残基。在CU2+、Ni2+、Zn2+等二价金属离子的存在下，IZ-HH呈现出一种展开结构，而形成三聚体盘绕结构。构建了融合蛋白（191）cpGFP(190)-IZ-HH，其中（191）cpGFP（190）插入到IZ-HH的第二和第三个a-螺旋之间。当CU2+和Zn2+离子存在时，表达（191）cpGFP(190)-IZ-HH的大肠杆菌细胞表现出强烈的荧光，表明CU2+和Zn2+离子穿过细胞膜，诱导191cpGFP190结构域的适当折叠。这种由金属离子响应线圈调节蛋白质功能的策略，应该适用于设计各种金属离子响应的非天然蛋白质，这些蛋白质在体外和体内都能起作用。
Coordination of metal ions significantly contributes to protein structures and functions. Here we constructed a fusion protein, consisting of a de novo designed, metal-ion-binding, trimeric coiled-coil and a circularly permutated green fluorescent protein (cpGFP), where the fluorescent emission from cpGFP was induced by metal ion coordination to the coiled-coil. A circularly permutated GFP, (191)cpGFP(190), was constructed by connecting the original N- and C-termini of GFP(uv) by a GGSGG linker and cleaving it between Asp(190) and Gly(191). The metal-ion-binding coiled-coil, IZ-HH, was designed to have three alpha-helical structures, with 12 His residues in the hydrophobic core of the coiled-coil structure. IZ-HH exhibited an unfolded structure, whereas it formed the trimeric coiled-coil structure in the presence of divalent metal ions, such as CU2+, Ni2+, or Zn2+. The fusion protein (191)cpGFP(190)-IZ-HH was constructed, in which (191)cpGFP(190) was inserted between the second and third a-helices of IZ-HH. Escherichia colicells, expressing (191)cpGFP(190)-IZ-HH, exhibited strong fluorescence when the CU2+ and Zn2+ ions were present in the medium, indicating that they passed through the cell membrane and induced the proper folding of the 191cpGFP190 domain. This strategy, in which protein function is regulated by a metal-ion-responsive coiled-coil, should be applicable to the design of various metal-ion-responsive, nonnatural proteins that work both in vitro and in vivo.