Improving a designed photocontrolled DNA-binding protein.
Improving a designed photocontrolled DNA-binding protein.
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DOI：
10.1021/bi101432p
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发表时间：
2011-02-22
期刊：
影响因子：
2.9
通讯作者：
Woolley, G. Andrew
中科院分区：
文献类型：
--
作者：
Fan, Helen Y.;Morgan, Stacy-Anne;Brechun, Katherine E.;Chen, Yih-Yang;Jaikaran, Anna S. I.;Woolley, G. Andrew
光控转录因子可能是探索转录过程在各种环境中的作用的强大工具。此前，我们基于 GCN4 的 bZIP 区域与来自 H. halophila 的光活性黄色蛋白之间的融合，设计了一种光控 DNA 结合蛋白 (Morgan et al., J. Mol. Biol. 2010, 399:94–112)。在这里，我们报告了一种基于结构的尝试，以提高用这种嵌合蛋白观察到的光开关程度。使用计算设计工具 PoPMuSiC 2.0、Rosetta、Eris 和 bCIPA，我们确定了一系列单点和多点突变，预计这些突变可以稳定蛋白质的折叠暗状态，从而增强光开关程度。虽然根据尿素变性研究判断，许多这些突变，特别是在 143 位引入疏水残基的突变，确实显着增强了暗态蛋白质稳定性，但暗态稳定性与光开关程度并不直接相关。相反，我们发现突变对光转换程度的影响与其对 DNA 结合减缓 PYP 光循环中 pB 到 pG 转变的程度的影响有关。一种突变体 K143F 导致光周期减慢约 10 倍，并且在 DNA 结合的表观 Kd 方面也表现出最大差异 - 照射后降低了 3.5 倍。由于该蛋白质家族与 DNA 结合的协同作用，表观 Kd 的这种变化导致辐射后结合 DNA 的分数增加 12 倍。结果强调了当前解决蛋白质设计实际问题的方法的优点和缺点，并提出了进一步改进设计的光控转录因子的策略。
Photo-controlled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photo-controlled DNA binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from H. halophila (Morgan et al., J. Mol. Biol. 2010, 399:94–112). Here we report a structure-based attempt to improve the degree of photoswitching observed with this chimeric protein. Using computational design tools PoPMuSiC 2.0, Rosetta, Eris and bCIPA we identified a series of single and multiple point mutations that were expected to stabilize the folded dark state of the protein and thereby enhance the degree of photoswitching. While a number of these mutations, particularly those that introduced a hydrophobic residue at position 143, did significantly enhance dark-state protein stability as judged by urea denaturation studies, dark-state stability did not correlate directly with the degree of photoswitching. Instead, the influence of mutations on the degree of photoswitching was found to be related to their effects on the degree to which DNA binding slowed the pB to pG transition in the PYP photocycle. One mutant, K143F, caused a ~10-fold slowing of the photocycle and also showed the largest difference in apparent Kd for DNA binding − 3.5-fold lower upon irradiation. This change in apparent Kd causes a 12-fold enhancement in fraction bound DNA upon irradiation due to the cooperativity of DNA binding by this family of proteins. The results highlight the strengths and weaknesses of current approaches to a practical problem in protein design as well as suggesting strategies for further improvement of designed photo-controlled transcription factors.
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影响因子：
2.7
作者：
Harigai, M;Yasuda, S;Kataoka, M
通讯作者：
Kataoka, M
影响因子：
3.4
作者：
Imamoto, Yasushi;Tatsumi, Sanae;Kataoka, Mikio
通讯作者：
Kataoka, Mikio
影响因子：
64.5
作者：
ELLENBERGER, TE;BRANDL, CJ;HARRISON, SC
通讯作者：
HARRISON, SC
DOI：
10.1038/nsb958
发表时间：
2003-08-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
Getzoff, ED;Gutwin, KN;Genick, UK
通讯作者：
Genick, UK
影响因子：
3.4
作者：
Hoersch, Daniel;Otto, Harald;Heyn, Maarten P.
通讯作者：
Heyn, Maarten P.