A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics.
A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics.
复制标题
基于细胞的四螺旋束蛋白 Rop 功能筛选：生物物理学组合实验的新工具。
DOI：
10.1093/protein/gzh010
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Regan,Lynne
中科院分区：
文献类型：
--
作者：
Magliery,ThomasJ;Regan,Lynne
Combinatorial methodologies have revolutionized studies in biomolecular function, but they have so far proven less useful for understanding macromolecular structure and stability. This is largely because of the difficulty of screening libraries of molecules for biophysical properties, and the difficulty of interpreting structural effects in complicated molecules. Here, we report a novel, robust, cell‐based screen for function of the four‐helix bundle protein, Rop. By expression of green fluorescent protein from a ColE1 plasmid, the screen reports the copy number of the plasmid, which is modulated inEscherichia coliby Rop. We have engineered the screen so that the fluorescent phenotype can correspond to either Rop activity or lack thereof. We have used the screen to demonstrate with systematically constructed Rop core variants that not all molecules that bind small stem–loop RNAsin vitroare activein vivo. Rop is well understood from structural work and systematic mutations, which makes it possible to construct rational, targeted libraries. This screen makes it possible to rapidly interrogate such libraries effectively for proper protein folding and stability. In addition to its intended utility for combinatorial experiments in biophysics, the screen will allow further dissection of the mechanism of Rop‐mediated plasmid copy number regulationin vivo.
