PROBING THE ROLES OF RESIDUES AT THE E-POSITION AND G-POSITION OF THE GCN4 LEUCINE-ZIPPER BY COMBINATORIAL MUTAGENESIS
PROBING THE ROLES OF RESIDUES AT THE E-POSITION AND G-POSITION OF THE GCN4 LEUCINE-ZIPPER BY COMBINATORIAL MUTAGENESIS
复制标题
DOI：
10.1002/pro.5560020701
复制
发表时间：
1993-07-01
期刊：
影响因子：
8
通讯作者：
SAUER, RT
中科院分区：
文献类型：
--
作者：
HU, JC;NEWELL, NE;SAUER, RT
组合诱变与字母表限于丙氨酸，谷氨酸，赖氨酸，和苏氨酸被用来探测涉及表面残基的相互作用的作用，稳定一个短的α-螺旋卷曲螺旋。将酿酒酵母转录因子GCN 4的亮氨酸拉链中的8个e和g位置处的残基随机化为λ阻遏物-亮氨酸拉链融合蛋白中的这4个残基，导致65，536种可能的残基组合。大约四分之三的这些组合允许稳定的卷曲螺旋的形成，如通过融合蛋白的DNA结合所测定的。为了理解活性差异的基础，对功能性和非功能性突变体进行测序，并应用统计学检验来鉴定结构/功能相关性。螺旋形成倾向和有利的亚基内和亚基间电荷-电荷相互作用与活性呈正相关。这些研究表明，在卷曲螺旋的e和g位置的表面侧链的身份有助于适度的稳定性，通过与以前的工作相比，然而，e和g位置远不如在a和d位置的残基关键，这形成了二聚体界面的疏水核心。
Combinatorial mutagenesis with an alphabet limited to alanine, glutamic acid, lysine, and threonine was used to probe the role of interactions involving surface residues in stabilizing a short alpha-helical coiled coil. The residues at eight e and g positions in the leucine zipper of the Saccharomyces cerevisiae transcription factor GCN4 were randomized to these four residues in a lambda repressor-leucine zipper fusion protein, resulting in 65,536 possible residue combinations. Roughly three-fourths of these combinations allowed stable coiled-coil formation as assayed by DNA binding by the fusion protein. To understand the basis for the activity differences, functional and nonfunctional mutants were sequenced and statistical tests were applied to identify structure/function correlations. Helix-forming propensity and favorable intrasubunit and intersubunit charge-charge interactions were positively correlated with activity. These studies suggest that the identities of surface side chains at the e and g positions of coiled coils contribute modestly to stability; by comparison with previous work, however, the e and g positions are far less critical than residues at the a and d positions, which form the hydrophobic core of the dimer interface.