Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.
Correlating in Vitro and in Vivo Activities of Light-Inducible Dimers: A Cellular Optogenetics Guide.
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DOI：
10.1021/acssynbio.5b00119
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发表时间：
2016-01-15
影响因子：
4.7
通讯作者：
Kuhlman B
中科院分区：
文献类型：
--
作者：
Hallett RA;Zimmerman SP;Yumerefendi H;Bear JE;Kuhlman B
光诱导二聚体是细胞光遗传学的有力工具，因为它们可以用来控制高空间和时间分辨率的蛋白质的定位和活性。尽管这种方法具有普遍性，但光诱导二聚体的应用并不总是简单的，因为在实现所需的生物活性之前，经常需要测试替代的二聚体系统和融合策略。由于对可用二聚体行为的生物物理/生化机制以及这与体内功能的相关性的不完全理解，这一过程进一步受到阻碍。为了更好地了解工程过程，我们检测了三种蓝光诱导二聚体变体(Cry2/CIB1、Ilid/SspB和LOVpep/ePDZ)的生物物理和生化特性，并将这些特性与体内共定位和功能分析相关联。我们发现，这些开关在暗态和亮态的结合亲和力有很大的不同，并且这些亲和力与体内各种测试中的活性变化有关，包括转录控制、细胞内定位研究和GTPase信号控制。此外，对于CRY2，我们观察到光诱导的同源寡聚变化可以对对替代融合策略敏感的活性有很大影响。
Light inducible dimers are powerful tools for cellular optogenetics as they can be used to control the localization and activity of proteins with high spatial and temporal resolution. Despite the generality of the approach, application of light inducible dimers is not always straightforward as it is frequently necessary to test alternative dimer systems and fusion strategies before the desired biological activity is achieved. This process is further hindered by an incomplete understanding of the biophysical/biochemical mechanisms by which available dimers behave and how this correlates to in vivo function. To better inform the engineering process we examined the biophysical and biochemical properties of three blue-light inducible dimer variants (Cryptochrome2 (CRY2)/CIB1, iLID/SspB, and LOVpep/ePDZ) and correlated these characteristics to in vivo co-localization and functional assays. We find that the switches vary dramatically in their dark-state and lit-state binding affinities, and that these affinities correlate with activity changes in a variety of in vivo assays including transcription control, intra-cellular localization studies and control of GTPase signaling. Additionally, for CRY2 we observe that light induced changes in homo-oligomerization can have large effects on activity that are sensitive to alternative fusion strategies.