Design and synthesis of a globin fold
Design and synthesis of a globin fold
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DOI：
10.1021/bi983006y
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发表时间：
1999-06-08
期刊：
影响因子：
2.9
通讯作者：
Nishikawa, K
中科院分区：
文献类型：
--
作者：
Isogai, Y;Ota, M;Nishikawa, K
我们提出了一种简单的方法来寻找可折叠成具有基于知识的 3D-1D 兼容性函数的所需结构的球状蛋白质的氨基酸序列。选择由 153 个氨基酸残基组成的抹香鲸肌红蛋白的不对称 α 螺旋单域结构作为设计目标。通过递归生成蛋白质 3D 图谱来搜索适合主链框架的最佳序列。血红素结合位点的设计是通过分别将 His64 和 His93 固定在远端和近端位置，并惩罚突出到空间中具有排斥功能的残基。根据最终的 3D 轮廓，通过用较小的残基替换一些碰撞残基，消除了收敛、自洽序列的计算机模型中侧链之间的明显碰撞。最终获得的序列与天然肌红蛋白有26%的序列共享。通过在大肠杆菌中表达合成基因获得了具有人工序列的设计的globin-1（DG1）。使用尺寸排阻色谱、圆二色光谱和溶液 X 射线散射进行的分析表明，DG1 折叠成单体、致密、高度螺旋和球状形式，其整体分子形状与水溶液中的目标结构相似。此外，它每个蛋白质分子结合一个血红素，表现出明确的光谱特性。根据X射线散射分析，DG1的回转半径确定为20.6埃，略大于天然apoMb的回转半径，并且在与血红素结合后降至19.5埃。然而，血红素结合的 DG1 不像天然球蛋白那样稳定地结合分子氧，这可能是由于 NMR 和变性实验中观察到的侧链结构的高度构象多样性所致。这些结果使人们深入了解序列选择与天然蛋白质实现生物学功能的结构独特性之间的关系。
We propose a simple method to find an amino acid sequence that is foldable into a globular protein with a desired structure based on a knowledge-based 3D-1D compatibility function. An asymmetric alpha-helical single-domain structure of sperm whale myoglobin consisting of 153 amino acid residues was chosen for the design target. The optimal sequence to fit the main-chain framework has been searched by recursive generation of the protein 3D profile. The heme-binding site was designed by fixing His64 and His93 at the distal and proximal positions, respectively, and by penalizing residues that protrude into the space with a repulsive function. The apparent bumps among side chains in the computer model of the converged, self-consistent sequence were removed by replacing some of the bumping residues with smaller ones according to the final 3D profile. The finally obtained sequence shares 26% of sequence with the natural myoglobin. The designed globin-1 (DG1) with the artificial sequence was obtained by expression of the synthetic gene in Escherichia coli. Analyses using size-exclusion chromatography, circular dichroism spectroscopy, and solution X-ray scattering showed that DG1 folds into a monomeric, compact, highly helical, and globular form with an overall molecular shape similar to the target structure in an aqueous solution. Furthermore, it binds a single heme per protein molecule, which exhibited well-defined spectroscopic properties. The radius of gyration of DG1 was determined to be 20.6 Angstrom, slightly larger than that of natural apoMb, and decreased to 19.5 Angstrom upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did notstably bind molecular oxygen as natural globins do, possibly due to high conformational diversity of side-chain structures observed in the NMR and denaturation experiments. These results give insight into the relationship between the sequence selection and the structural uniqueness of natural proteins to achieve biological functions.