Computational design and characterization of a monomeric helical dinuclear metalloprotein
Computational design and characterization of a monomeric helical dinuclear metalloprotein
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DOI：
10.1016/j.jmb.2003.10.004
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发表时间：
2003-12-12
影响因子：
5.6
通讯作者：
Saven, JG
中科院分区：
文献类型：
--
作者：
Calhoun, JR;Kono, H;Saven, JG
双铁蛋白的从头设计是理解这一复杂的金属酶家族功能多样性的重要一步。先前对双铁（DF）蛋白的设计产生了具有晶体学上明确结构和活性位点几何形状的四聚体和二聚体四螺旋束。在此，介绍了DFsc（一种含114个残基的单体四螺旋束）的设计与表征。主链是利用先前的寡聚体结构和适当的螺旋间转角进行建模的。26个残基的特性是预先确定的，包括活性位点中的一级和二级配体、与活性位点可及性有关的残基以及螺旋2和3之间的γβγβ转角。其余88个氨基酸残基是利用基于近期蛋白质序列统计理论的统计计算机辅助设计确定的。该理论不是对序列进行抽样，而是直接提供位点特异性氨基酸概率，然后用于指导序列设计。所得序列（DFsc）在大肠杆菌中表达良好且溶解性高。沉降研究证实该蛋白质在溶液中为单体。圆二色性光谱与目标结构的螺旋含量相符。该蛋白质在脱辅基形式和全酶形式下均具有结构，且金属结合形式表现出更高的稳定性。DFsc能以化学计量方式结合多种二价金属离子，包括锌（II）、钴（II）、铁（II）和锰（II），亲和力为微摩尔级别。脱辅基蛋白质和锌（II）结合蛋白质的N - 15 HSQC NMR光谱显示出良好的分散性，且有金属结合时发生显著结构变化的证据。因此，DFsc是完全从头设计的一个实例，在设计过程中确定了主链结构、活性和序列。（C）2003爱思唯尔有限公司。保留所有权利。
The de novo design of di-iron proteins is an important step towards understanding the diversity of function among this complex family of metallo-enzymes. Previous designs of due ferro (DF) proteins have resulted in tetrameric and dimeric four-helix bundles having crystallographically well-defined structures and active-site geometries. Here, the design and characterization of DFsc, a 114 residue monomeric four-helix bundle, is presented. The backbone was modeled using previous oligomeric structures and appropriate inter-helical turns. The identities of 26 residues were predetermined, including the primary and secondary ligands in the active site, residues involved in active site accessibility and the gammabetagammabeta turn between helices 2 and 3. The remaining 88 amino acid residues were determined using statistical computer aided design, which is based, upon a recent statistical theory of protein sequences. Rather than sampling sequences, the theory directly provides the site-specific amino acid probabilities, which are then used to guide sequence design. The resulting sequence (DFsc) expresses well in Escherichia coli and is highly soluble. Sedimentation studies confirm that the protein is monomeric in solution. Circular dichroism spectra are consistent with the helical content of the target structure. The protein is structured in both the apo and the holo forms, with the metal-bound form exhibiting increased stability., DFsc stoichiometrically binds a variety of divalent metal ions, including Zn(II), Co(II), Fe(II), and Mn(II), with micromolar affinities. N-15 HSQC NMR spectra of both the apo and Zn(II) proteins reveal excellent dispersion with evidence of a significant structural change upon metal binding. DFsc is then a realization of complete de novo design, where backbone structure, activity, and sequence are specified in the design process. (C) 2003 Elsevier Ltd. All rights reserved.