Modeling Fluorescent and Tracer Proteins (GFP and luciferase)

荧光和示踪蛋白（GFP 和荧光素酶）建模

• 批准号: 7070149
• 负责人: MARC ZIMMER
• 金额: $ 14.95万
• 依托单位: CONNECTICUT COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-04-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7070149
• 关键词: catalyst; chemical models; chemical registry /resource; chemical structure; cheminformatics; chromophore; computational biology; computer program /software; computer simulation; entactin; fluorescent dye /probe; gene expression; green fluorescent proteins; luciferin monooxygenase; mathematical model; model design /development; molecular dynamics; photochemistry; protein localization

DESCRIPTION (provided by applicant): Green fluorescent protein (GFP) and firefly luciferase(luc) are commonly used tracer proteins. They have applications in many areas of molecular biology, genetics, and cell biology where they have been used as fusion tags to visualize dynamic cellular events and to monitor protein localization, and as reporter genes to monitor gene expression. Both GFP and luc are commonly used in medicine. For example, to visualize tumor metastasis and angiogenesis, to monitor the spread of herpes simplex virus, to get a better understanding of the role of beta cells in diabetes development, and to examine the misfolding of proteins in early onset dystonia. They have also been used in the fight against bioterrorism e.g. to detect anthrax. I propose using computational methods to: Examine the autocatalytic chromophore formation in GFP. What is the structure of the precyclized form of wild-type GFP? What is the role of Arg96? The answers to these questions will hopefully help in designing mutants that form the chromophore more efficiently. Computationally design fluorescent nidogen mutants. Nidogen has a very similar tertiary structure to GFP, but it does not form a chromophore. What is the smallest set of mutations that can lead to chromophore formation in nidogen? * The effect of the protein matrix on chromophore rotation. The GFP chromophore only fluoresces when GFP is in its native conformation this is because the protein matrix restricts chromophore rotation. I would like to use molecular dynamics simulations to examine the low energy conformations available to a freely rotating chromophore in all the GFP and GFP-like proteins in the protein database. This includes chromophoric non- fluorescent proteins. This work will help us understand the photochemistry of GFP, especially the light and dark states. Use homology and conformational searching methods to generate the active conformations of luc from the crystal structure of its inactive form and the known active conformations of some of the members in its protein superfamily. The results should lead to a better understanding of the synthetase and monoxygenase functions of luc.

描述（由申请人提供）：绿色荧光蛋白（GFP）和萤火虫荧光素酶（luc）是常用的示踪蛋白。它们在分子生物学、遗传学和细胞生物学的许多领域都有应用，在这些领域中，它们已被用作融合标签以可视化动态细胞事件并监测蛋白质定位，以及用作报告基因以监测基因表达。GFP和luc都是医学中常用的。例如，可视化肿瘤转移和血管生成，监测单纯疱疹病毒的传播，更好地了解β细胞在糖尿病发展中的作用，以及检查早发性肌张力障碍中蛋白质的错误折叠。它们还被用于打击生物恐怖主义，例如检测炭疽。我建议使用计算方法：检查自催化的发色团形成的绿色荧光蛋白。野生型GFP的预环化形式的结构是什么？Arg96的作用是什么？这些问题的答案将有望有助于设计更有效地形成发色团的突变体。计算设计荧光巢蛋白突变体。巢蛋白具有与GFP非常相似的三级结构，但它不形成发色团。哪一组最小的突变可以导致巢蛋白中发色团的形成？* 蛋白质基质对发色团旋转的影响。GFP发色团仅在GFP处于其天然构象时发荧光，这是因为蛋白质基质限制发色团旋转。我想用分子动力学模拟来研究低能量构象可自由旋转的发色团在所有的绿色荧光蛋白和绿色荧光蛋白样蛋白的蛋白质数据库。这包括发色非荧光蛋白。这项工作将有助于我们了解绿色荧光蛋白的光化学，特别是光和暗状态。使用同源性和构象搜索方法，从luc的非活性形式的晶体结构和其蛋白超家族中某些成员的已知活性构象生成luc的活性构象。这些结果将有助于我们更好地理解luc的合成酶和单加氧酶功能。