Czupryn M, Bennett F, Dube J, Grant K, Scoble H, Sookdeo H, McCoy J M
Genetics Institute Inc., Cambridge, Massachusetts 02140, USA.
Ann N Y Acad Sci. 1995 Jul 21;762:152-64. doi: 10.1111/j.1749-6632.1995.tb32323.x.
We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.
我们通过丙氨酸扫描诱变确定了人白细胞介素-11（hIL-11）的功能重要区域。在大肠杆菌中作为硫氧还蛋白融合蛋白产生了总共61种hIL-11的突变形式，并在小鼠T10浆细胞瘤增殖试验中进行了测试。在hIL-11 C末端附近的几个位置进行的突变导致生物活性大幅降低。此外，该区域的许多其他突变影响了蛋白质折叠或稳定性。两种效应在一级序列上都表现出特征性的周期性，这表明hIL-11 C末端附近的残基呈现螺旋构象。在hIL-11 N末端附近进行的突变也表现出生物活性降低，尽管未观察到对蛋白质折叠或稳定性的影响。活性降低的N末端突变也呈现出暗示螺旋构象的周期性。我们之前基于物理研究、选择性化学修饰、内含子/外显子边界位置、有限蛋白酶解实验和定点诱变提出了hIL-11结构的四螺旋束拓扑结构。我们在此报告的丙氨酸扫描诱变数据为该模型提供了额外支持。
