The Combinatorial Design of Protein Molecular Switches

蛋白质分子开关的组合设计

• 批准号: 6908877
• 负责人: MARC A OSTERMEIER
• 金额: $ 27.35万
• 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/6908877
• 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）： 蛋白质分子开关在功能上将外部信号（例如配体结合）与功能息息相关。分子开关具有多种潜在的与健康相关的应用，包括基因转录的调节，细胞信号通路的调节，靶向药物输送，药物转运，有条件活性的有毒毒物蛋白的创建以及分子生物传感器的创建。尽管具有很大的潜力，但蛋白质开关的创建并未得到广泛的探索，部分原因是其工程的一般策略很少。使用将生物学，化学和工程方法整合的组合方法，分子开关将通过一种新型策略进行设计，即使用“原理证明”实验中的模型蛋白的“组合域插入”策略。在组合结构域插入中，将两个基因融合，使一个基因在另一个基因中随机插入。在选择的模型系统中，基因A代码为在信号存在下经历构象变化的结合蛋白（例如配体结合）。基因B代码要控制蛋白质（例如酶）。从这些文库中，将确定融合蛋白在功能上逐步划出两个域的功能（例如配体结合调节酶的活性）。假设该功能耦合是由蛋白A的配体依赖性构象/稳定性变化产生的，该蛋白A A的影响B。获得的代表性开关将在动力学和结构上表征。通过组合方法进行的系统分析以及创建的开关的生化和结构表征，将开发开关机理的模型，并通过实验进行测试，以阐明一般原理的目的进行测试，这些原理可以应用于生物医学应用的分子开关的创建。