STRUCTURAL CONSEQUENCES OF REDUCTIVE METHYLATION OF LYSINE RESIDUES IN HEN EGG-WHITE LYSOZYME - AN X-RAY-ANALYSIS AT 1.8-ANGSTROM RESOLUTION
STRUCTURAL CONSEQUENCES OF REDUCTIVE METHYLATION OF LYSINE RESIDUES IN HEN EGG-WHITE LYSOZYME - AN X-RAY-ANALYSIS AT 1.8-ANGSTROM RESOLUTION
复制标题
DOI：
10.1021/bi00088a041
复制
发表时间：
1993-09-21
期刊：
影响因子：
2.9
通讯作者：
RAYMENT, I
中科院分区：
文献类型：
--
作者：
RYPNIEWSKI, WR;HOLDEN, HM;RAYMENT, I
蛋白质的化学修饰已经并将继续成为研究蛋白质结构和功能的重要生化工具。一种此类方法是赖氨酸残基的还原甲基化。为了解决这种甲基化对蛋白质结晶和结构特性的影响，确定了鸡蛋清溶菌酶的三维结构，其中所有赖氨酸残基都已被烷基化，并精炼至标称分辨率为1.8埃，晶体学R因子为17.3%。研究中使用的晶体由 1.5-1.8 M MgSO4 和 50 mM Tris 在 pH 8.0 下生长而成，属于空间群 P2(1)2(1)2，晶胞尺寸为 a = 30.6 埃、b = 56.3 埃、c = 73.2 埃，每个不对称单元有一个分子。在通常用于鸡蛋清蛋白的条件下不可能生长修饰的溶菌酶晶体。总体而言，天然溶菌酶和修饰蛋白的三维结构非常相似，只有两个表面环有显着程度的差异。具体来说，这两种形式的蛋白质的 α-碳位置（不包括表面环）以 0.40 埃的均方根值叠加。在修饰和未修饰形式的溶菌酶之间观察到的结构变化的程度与当相同的蛋白质结构在两种不同的晶格中溶解时所观察到的结构变化的程度相似。因此，赖氨酸残基的甲基化导致的结构扰动非常小，但会对蛋白质的结晶特性产生巨大影响。如本文所述，该技术对于获得肌球蛋白亚片段 1 的 X 射线质量晶体绝对关键，因此可能被证明对于迄今为止难以形成有序阵列的其他蛋白质的结晶是有价值的。
Chemical modification of proteins has been and continues to be an important biochemical tool for the study of protein structure and function. One such type of approach has been the reductive methylation of lysine residues. In order to address the consequences of such methylation on the crystallization and structural properties of a protein, the three-dimensional structure of hen egg white lysozyme in which all lysine residues have been alkylated has been determined and refined to a nominal resolution of 1.8 angstrom and a crystallographic R factor of 17.3%. Crystals used in the investigation were grown from 1.5-1.8 M MgSO4 and 50 mM Tris at pH 8.0 and belonged to, the space group P2(1)2(1)2, with unit cell dimensions of a = 30.6 angstrom, b = 56.3 angstrom, c = 73.2 angstrom, and one molecule per asymmetric unit. It was not possible to grow crystals of the modified lysozyme under the conditions normally employed for the hen egg white protein. Overall, the three-dimensional structures of the native lysozyme and the modified protein are very similar with only two surface loops differing to any significant extent. Specifically, the positions of the alpha-carbons for these two forms of the protein, excluding the surface loops, superimpose with a root-mean-square value of 0.40 angstrom. The magnitude of the structural changes observed between the modified and unmodified forms of lysozyme is similar to that seen when an identical protein structure is solved in two different crystalline lattices. Consequently, the methylation of lysine residues results in very little structural perturbations but can produce enormous effects on the crystallization properties of a protein. As described here, this technique was absolutely critical for obtaining X-ray quality crystals of myosin subfragment 1 and thus may prove to be valuable in the crystallization of other proteins that have so far resisted forming ordered arrays.