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Improving a Designed Photocontrolled DNA-Binding Protein

机译：改善设计的光控DNA结合蛋白

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Photocontrolled transcription factors could be powerful tools for probing the roles of transcriptional processes in a variety of settings. Previously, we designed a photocontrolled DNA-binding protein based on a fusion between the bZIP region of GCN4 and photoactive yellow protein from Halorhodospira halophila [Morgan, S. A., et al. (2010) J. Mol. Biol. 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 an similar to 10-fold slowing of the photocycle and also showed the largest difference in the apparent K-d for DNA binding, 3.5-fold lower, upon irradiation. This change in the apparent K-d causes a 12-fold enhancement in the 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 and suggest strategies for further improvement of designed photocontrolled transcription factors.
机译：光控转录因子可能是在各种环境中探索转录过程作用的强大工具。以前，我们基于GCN4的bZIP区域和嗜盐嗜盐气单胞菌的光敏黄色蛋白[Morgan，S. A.，等。 （2010）J.Mol。生物学399，94-112]。在这里我们报告了基于结构的尝试，以改善这种嵌合蛋白观察到的光开关的程度。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，这些突变有望稳定蛋白质的折叠暗态，从而增强光开关的程度。尽管许多突变，特别是那些在143位引入疏水残基的突变，确实通过尿素变性研究判断确实增强了暗态蛋白质的稳定性，但暗态稳定性与光开关的程度并不直接相关。取而代之的是，发现突变对光开关程度的影响与其对DNA结合在PYP光周期中减慢pB到pG过渡程度的影响有关。一个突变体K143F导致光循环的减慢幅度接近10倍，并且在辐照后与DNA结合的表观K-d差异最大，降低了3.5倍。表观K-d的这种变化由于该蛋白质家族结合DNA的协同作用，导致照射后结合的DNA组分增加了12倍。结果突出了目前解决蛋白质设计中实际问题的方法的优缺点，并提出了进一步改善设计的光控转录因子的策略。

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