The Combinatorial Design of Protein Molecular Switches

蛋白质分子开关的组合设计

• 批准号: 6684914
• 负责人: MARC A OSTERMEIER
• 金额: $ 26.73万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6684914
• 关键词: allosteric site; beta lactamase; binding proteins; bioengineering /biomedical engineering; chemical models; circular dichroism; combinatorial chemistry; conformation; enzyme activity; fusion gene; green fluorescent proteins; ligands; maltose; model design /development; molecular dynamics; peptide library; protein protein interaction; protein purification; protein sequence; protein structure function; proteomics; site directed mutagenesis; transposon /insertion element

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.

描述（由申请人提供）： 蛋白质分子开关将外部信号（例如配体结合）与功能性耦合。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、药物运输、条件活性有毒蛋白的创建以及分子生物传感器的创建。尽管蛋白质开关具有巨大的潜力，但其创建尚未得到广泛探索，部分原因是缺乏其工程的通用策略。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称为“组合域插入”的新策略在“原理验证”实验中使用模型蛋白来设计。在组合结构域插入中，两个基因被融合，使得一个基因随机插入另一个基因中。在所选的模型系统中，基因 A 编码结合蛋白，该结合蛋白在信号（例如配体结合）存在下发生构象变化。基因 B 编码要控制的蛋白质（例如酶）。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）。假设功能耦合是由影响蛋白质 B 活性的蛋白质 A 中配体依赖性构象/稳定性变化引起的。所获得的代表性开关将在动力学和结构上进行表征。通过组合方法提供的系统分析以及所创建的开关的生化和结构表征，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生物医学应用分子开关的一般原理。