Computational Investigations of Monomeric Variants of Red Fluorescent Proteins

红色荧光蛋白单体变体的计算研究

• 批准号: 8306851
• 负责人: Prem P Chapagain
• 金额: $ 10.88万
• 依托单位: FLORIDA INTERNATIONAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-25 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8306851
• 关键词: Active Biological Transport; Active Sites; Amino Acid Substitution; Amino Acids; Biochemical Markers; Biochemical Pathway; Cells; Cellular biology; Computer Simulation; Diffusion; Disease; DsRed; Effectiveness; Future; Goals; Green Fluorescent Proteins; Individual; Investigation; Light; Location; Membrane Proteins; Modeling; Modification; Molecular; Molecular Biology; Monitor; Oxygen; Pathway interactions; Permeability; Photobleaching; Play; Preparation; Process; Property; Protein Region; Proteins; Reaction; Reactive Oxygen Species; Research; Role; Site; Site-Directed Mutagenesis; Solvents; Structure; Structure-Activity Relationship; Surface; System; Time; Variant; Work; beta barrel; chromophore; computer studies; design; flexibility; improved; in vivo; interest; molecular dynamics; oxygen transport; passive transport; prevent; protein aggregation; red fluorescent protein; tool

DESCRIPTION (provided by applicant): The goal of this research is to improve the fluorescent properties of an important class of fluorescent proteins. Fluorescent proteins are extremely valuable biochemical markers in molecular and cell biology that allow monitoring of cellular biology on a molecular level. Fluorescent proteins are used to elucidate the biochemical pathways of healthy cells, and uncover the molecular problems that cause diseases. Red fluorescent proteins (RFPs) are highly desirable for in vivo applications because they absorb and emit light in the red region of the spectrum where cellular autofluorescence is low. Naturally occurring RFPs are polymeric, which makes them unsuitable for use in tagging purposes. Structural modifications to these RFPs have produced monomeric RFP variants, but at present their usefulness is limited because they are not photostable. We hypothesize that due to the compromised structural integrity of monomeric RFPs, the chromophore is not well protected from attack by molecular oxygen. Computational investigations of structure-function relationships will allow the design of protein- chromophore monomeric RFP variants that are impermeable to oxygen for extended periods of time. These computationally designed monomeric RFP variants will assist experimentalists in preparing RFPs with chromophores that are better protected and with substantially improved photostability. This work will also develop tools for future computational design of monomeric RFPs with protein-chromophore combinations with improved brightness. The proposed research has three specific Aims: (1)Parameterization of force fields for various chromophores to use in computational studies (2)Investigation of the structural integrity and fluctuations of the proteins of monomeric variants of RFPs to identify key regions where oxygen can enter and attack the chromophore. (3)Identify protein regions with specific amino acids that can temporarily host oxygen and release it at the appropriate time to actively assist the debilitating transport of oxygen to the interior. Identifying these regions will permit a systematic computational-experimental strategy of amino acid substitution to create more protective proteins, and to modify the protein-chromophore interactions to enhance the fluorescent properties of the system.

描述（由申请人提供）：这项研究的目的是改善重要类别的荧光蛋白的荧光性能。荧光蛋白是分子和细胞生物学中极有价值的生化标记，可在分子水平上监测细胞生物学。荧光蛋白用于阐明健康细胞的生化途径，并发现引起疾病的分子问题。红色荧光蛋白（RFP）非常需要体内应用，因为它们在细胞自动荧光较低的光谱中吸收并发出光。自然发生的RFP是聚合物，这使得它们不适合用于标记目的。这些RFP的结构修饰产生了单体RFP变体，但目前它们的实用性受到限制，因为它们不可光稳定。我们假设由于单体RFP的结构完整性受损，发色团不能很好地保护分子氧的攻击。结构功能关系的计算研究将允许设计蛋白质 - 发色团单体RFP变体，这些变体长时间不可渗透氧气。这些计算设计的单体RFP变体将有助于实验者制备具有更好保护并具有显着改善光稳定性的发色团的RFP。这项工作还将开发工具，用于以蛋白质 - 肉眼组合组合和亮度改善的蛋白质 - 肉眼组合的单粒RFP的未来计算设计。拟议的研究具有三个具体目的：（1）各种发色团的力场参数化用于计算研究（2）研究RFPS单体变体蛋白质的结构完整性和波动，以识别氧气可以进入和攻击发色体的关键区域。 （3）鉴定具有特定氨基酸的蛋白质区域，这些氨基酸可以暂时托管氧气并在适当的时间释放它，以积极帮助氧气向内部的衰弱。确定这些区域将允许氨基酸取代的系统计算实验策略创建更多的保护蛋白，并修改蛋白质 - 肉眼相互作用，以增强系统的荧光性能。