The Combinatorial Design of Protein Molecular Switches

描述(由申请人提供)： 蛋白质分子开关在功能上将外部信号(如配体结合)与功能相结合。分子开关具有广泛的潜在的健康相关应用，包括调节基因转录、调节细胞信号通路、靶向药物输送、药物转运、产生有条件活性的有毒蛋白以及制造分子生物传感器。尽管它们具有巨大的潜力，但蛋白质开关的创造还没有得到广泛的探索，部分原因是缺乏对它们的工程的一般策略。利用结合了生物、化学和工程方法的组合方法，分子开关将通过一种名为组合结构域插入的新策略进行工程设计，该策略使用模型蛋白质进行原理验证实验。在组合域插入中，两个基因融合在一起，其中一个随机插入另一个。在所选择的模型系统中，基因A编码结合蛋白，该结合蛋白在信号(例如，配体结合)的存在下经历构象变化。基因B编码一种受控制的蛋白质(例如一种酶)。从这些文库中，将鉴定出在功能上耦合两个结构域功能的融合蛋白(例如，配体结合调节酶的活性)。这种功能偶联被认为是由于蛋白质A的配体依赖的构象/稳定性变化导致的，这些变化影响了蛋白质B的活性。获得的代表性开关将被动力学和结构表征。通过组合方法提供的系统分析以及创建的开关的生化和结构特征，将开发开关机制的模型并进行实验测试，目的是阐明可应用于生物医学应用的分子开关的一般原理。

DESCRIPTION (provided by applicant): Protein molecular switches functionally couple external signals (such as ligand binding) to functionality. Molecular switches have a wide variety of potential health related applications including the regulation of gene transcription, the modulation of cell signaling pathways, targeted drug delivery, drug transport, the creation of conditionally active toxic proteins, and the creation of molecular biosensors. Despite their great potential, the creation of protein switches has not been extensively explored, in part due to the paucity of general strategies for their engineering. Using combinatorial methods that integrate biological, chemical and engineering approaches, molecular switches will be engineered by a novel strategy called 'combinatorial domain insertion' using model proteins in 'proof-of-principle' experiments. In combinatorial domain insertion, two genes are fused such that one is randomly inserted within the other. In the model system chosen, Gene A codes for a binding protein that undergoes a conformational change in the presence of a signal (e.g. ligand binding). Gene B codes for a protein to be controlled (e.g. an enzyme). From these libraries, fusion proteins will be identified that functionally couple the two domains' functions (e.g. ligand binding modulates the enzyme's activity). The functional coupling is hypothesized to result from ligand-dependent conformational/stability changes in protein A that affect the activity of protein B. Representative switches obtained will be kinetically and structurally characterized. Through the systematic analysis afforded by a combinatorial approach and the biochemical and structural characterization of the switches created, models of the mechanism of switching will be developed and tested experimentally with the goal of elucidating general) principles that can be applied to the creation of molecular switches for biomedical applications.