Isogai Y, Ota M, Fujisawa T, Izuno H, Mukai M, Nakamura H, Iizuka T, Nishikawa K
The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Biochemistry. 1999 Jun 8;38(23):7431-43. doi: 10.1021/bi983006y.
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 A, slightly larger than that of natural apoMb, and decreased to 19.5 A upon heme binding based on X-ray scattering analysis. However, the heme-bound DG1 did not stably 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.
我们提出了一种简单的方法，基于一种基于知识的3D-1D兼容性函数，找到一个可折叠成具有所需结构的球状蛋白质的氨基酸序列。选择了由153个氨基酸残基组成的抹香鲸肌红蛋白的不对称α-螺旋单结构域结构作为设计目标。通过递归生成蛋白质3D轮廓来搜索适合主链框架的最佳序列。通过分别将His64和His93固定在远端和近端位置，并通过用排斥函数惩罚突入空间的残基来设计血红素结合位点。根据最终的3D轮廓，用较小的残基替换一些碰撞残基，消除了收敛的、自洽序列的计算机模型中侧链之间明显的凸起。最终获得的序列与天然肌红蛋白有26%的序列相同。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球蛋白-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在水溶液中折叠成单体、紧凑、高度螺旋的球状形式，其整体分子形状与目标结构相似。此外，每个蛋白质分子结合一个血红素，表现出明确的光谱特性。基于X射线散射分析，DG1的回转半径确定为20.6 Å，略大于天然脱辅基肌红蛋白，血红素结合后降至19.5 Å。然而，与天然球蛋白不同，血红素结合的DG1不能稳定地结合分子氧，这可能是由于在核磁共振和变性实验中观察到侧链结构的高构象多样性。这些结果深入了解了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。
