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）与功能性偶联。分子开关具有多种潜在的健康相关应用，包括基因转录的调节、细胞信号传导途径的调节、靶向药物递送、药物转运、条件活性毒性蛋白的产生以及分子生物传感器的产生。尽管它们具有巨大的潜力，但蛋白质开关的创建尚未被广泛探索，部分原因是缺乏通用的工程策略。使用整合生物，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，使用模型蛋白质进行“原理验证”实验。在组合结构域插入中，两个基因融合，使得一个基因随机插入另一个基因中。在所选择的模型系统中，基因A编码结合蛋白，其在信号存在下经历构象变化（例如配体结合）。基因B编码要控制的蛋白质（例如酶）。从这些文库中，将鉴定功能性地偶联两个结构域的功能（例如，配体结合调节酶的活性）的融合蛋白。假设功能偶联是由蛋白A中影响蛋白B活性的配体依赖性构象/稳定性变化引起的。将对获得的代表性开关进行动力学和结构表征。通过由组合方法提供的系统分析以及所创建的开关的生物化学和结构表征，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创建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.