Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor
Computational de novo design and characterization of a four-helix bundle protein that selectively binds a nonbiological cofactor
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DOI：
10.1021/ja044129a
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发表时间：
2005-02-09
影响因子：
15
通讯作者：
DeGrado, WF
中科院分区：
文献类型：
--
作者：
Cochran, FV;Wu, SP;DeGrado, WF
我们报告了四螺旋束蛋白的完整从头设计，该蛋白选择性结合非生物 DPP−Fe(III) 金属卟啉辅因子（DPP−Fe(III) = 5, 15-Di[(4-羧基亚甲基氧基)苯基]卟啉铁(III)）。构建了四聚体 D2 对称骨架支架，通过双 (His) 配位封装两个 DPP−Fe(III) 单元。借助统计计算设计算法 SCADS 确定完整序列。 34个残基的肽是化学合成的。 UV-vis 和 CD 光谱、尺寸排阻色谱和分析超速离心表明，该肽在结合 DPP-Fe(III) 后经历了从主要是无规卷曲单体到 α-螺旋四聚体的转变。 EPR 光谱研究表明，轴向咪唑配体以垂直方式取向，如设计中包含的第二壳层相互作用所定义的。组装蛋白质的 1-D1H NMR 谱显示出内部填充良好的特征。组装的蛋白质具有与含有血红素辅因子的结构相似的系统不同的功能性氧化还原特性。设计的肽表现出显着的辅因子选择性，对天然血红素辅因子的结合亲和力明显较弱。这些发现为选择性地将更精细的辅助因子纳入设计的支架中以构建分子明确的纳米级材料开辟了道路。
We report the complete de novo design of a four-helix bundle protein that selectively binds the nonbiological DPP−Fe(III) metalloporphyrin cofactor (DPP−Fe(III) = 5, 15-Di[(4-carboxymethyleneoxy)phenyl]porphinato iron(III)). A tetrameric,D2-symmetric backbone scaffold was constructed to encapsulate two DPP−Fe(III) units through bis(His) coordination. The complete sequence was determined with the aid of the statistical computational design algorithm SCADS. The 34-residue peptide was chemically synthesized. UV−vis and CD spectroscopy, size-exclusion chromatography, and analytical ultracentrifugation indicated the peptide undergoes a transition from a predominantly random coil monomer to an α-helical tetramer upon binding DPP−Fe(III). EPR spectroscopy studies indicated the axial imidazole ligands were oriented in a perpendicular fashion, as defined by second-shell interactions that were included in the design. The 1-D1H NMR spectrum of the assembled protein displayed features of a well-packed interior. The assembled protein possessed functional redox properties different from those of structurally similar systems containing the heme cofactor. The designed peptide demonstrated remarkable cofactor selectivity with a significantly weaker binding affinity for the natural heme cofactor. These findings open a path for the selective incorporation of more elaborate cofactors into designed scaffolds for constructing molecularly well-defined nanoscale materials.