Design of λ Cro fold:: Solution structure of a monomeric variant of the de novo protein
Design of λ Cro fold:: Solution structure of a monomeric variant of the de novo protein
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DOI：
10.1016/j.jmb.2005.10.005
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发表时间：
2005-12-09
影响因子：
5.6
通讯作者：
Ota, M
中科院分区：
文献类型：
--
作者：
Isogai, Y;Ito, Y;Ota, M
经典的DNA结合蛋白之一——噬菌体λ Cro，可形成由α - 螺旋和β - 折叠构成独特折叠结构的同型二聚体。我们通过基于知识的结构 - 序列兼容性函数，利用模拟退火算法，从计算角度设计了一段由60个氨基酸残基组成的人工序列，以稳定λ Cro二聚体的骨架三级结构。所设计的氨基酸序列与天然λ Cro的序列有25% 的一致性，并且保留了苯丙氨酸58（Phe58），该氨基酸对于λ Cro稳定折叠结构的形成至关重要。我们合成了所设计的二聚体蛋白及其单体变体（通过在C端区域插入β - 发夹序列以防止二聚化而重新设计），并对其进行生化特性分析，结果表明它们折叠良好。所设计的蛋白在很宽的蛋白浓度范围内都以单体形式存在，并且通过核磁共振波谱法测定了其溶液结构。解析出的结构与天然λ Cro单体变体的结构相似，多肽骨架的均方根偏差为2.1埃，且具有紧密堆积的蛋白核心。因此，我们基于知识的函数提供了氨基酸序列与蛋白质结构之间近似但关键的关系，对于寻找可折叠成给定目标结构的新序列很有用处。
One of the classical DNA-binding proteins, bacteriophage lambda Cro, forms a homodimer with a unique fold of alpha-helices and beta-sheets. We have computationally designed an artificial sequence of 60 amino acid residues to stabilize the backbone tertiary structure of the lambda Cro dimer by simulated annealing using knowledge-based structure-sequence compatibility functions. The designed amino acid sequence has 25% identity with that of natural lambda Cro and preserves Phe58, which is important for formation of the stably folded structure of lambda Cro. The designed dimer protein and its monomeric variant, which was redesigned by the insertion of a beta-hairpin sequence at the C-terminal region to prevent dimerization, were synthesized and biochemically characterized to be well folded. The designed protein was monomeric under a wide range of protein concentrations and its solution structure was determined by NMR spectroscopy. The solved structure is similar to that of a monomeric variant of natural lambda Cro with a root-mean-square deviation of the polypeptide backbones at 2.1 angstrom and has a well-packed protein core. Thus, our knowledge-based functions provide approximate but essential relationships between amino acid sequences and protein structures, and are useful for finding novel sequences that are foldable into a given target structure.