Development of a Photo-cleavable Agent for Reversible Protein Dimerization

描述(申请人提供)：该提案寻求开发一种新的方法来控制细胞内工程蛋白的活性。化学二聚剂的使用 例如Rapalog作为一种激活细胞表面受体或通过使细胞内的蛋白质交联来使其失效的方法是非常有价值的。我们试图创造一种化学诱导的二聚化试剂(CID)，它可以被光切割，从而使二聚化反应迅速可逆。这样的分子需要在细胞中缺乏内源性结合伙伴，容易被膜所取代，没有毒副作用，并且可以被不会造成细胞损伤或干扰标准成像方法的波长的光有效地切割。我们建议对候选的可光裂解的CID进行表征，以确定其动力学和浓度依赖性，从而验证其在细胞培养中的适用性。我们提出了优化其亲和力和有效性的方法，并建议在轴突运输的“分裂动蛋白”实验中测试该试剂，以确定它是否可以用于在活神经元中将货物与其顺行运动迅速分离。

DESCRIPTION (provided by applicant): This proposal seeks to develop a novel method for controlling the activity of engineered proteins within cells. The use of chemical dimerizing agents such as rapalog have been very valuable as a means of activating cell surface receptors or disabling proteins in a cell by crosslinking them. We seek to create a Chemically-Induced Dimerization agent (CID) that is cleaved by light, thereby making the dimerization rapidly reversible. Such a molecule needs to lack endogenous binding partners in cells, be readily membrane permeant, have no toxic side effects, and be efficiently cleaved by light of a wavelength that will not cause cellular damage or interfere with standard imaging methods. We propose to characterize a candidate photocleavable CID to determine its kinetics and concentration dependence and thereby validate its suitability for work in cells in culture. We propose methods to optimize its affinity and efficacy, and we propose to test the reagent in a "split kinesin" assay of axonal transport to determine if it can be used to rapidly uncouple a cargo from its anterograde motor in a live neuron.