Computational Investigations of Monomeric Variants of Red Fluorescent Proteins

描述(申请人提供)：这项研究的目标是改善一类重要的荧光蛋白的荧光性质。荧光蛋白是分子和细胞生物学中非常有价值的生化标记，可以在分子水平上监测细胞生物学。荧光蛋白被用来阐明健康细胞的生化途径，并揭示导致疾病的分子问题。红色荧光蛋白(RFP)在体内的应用是非常理想的，因为它们在光谱的红色区域吸收和发射光，那里的细胞自身荧光很低。自然产生的RFP是聚合的，这使得它们不适合用于标记目的。对这些RFP的结构修饰已经产生了单体RFP变体，但目前它们的用途受到限制，因为它们不是光稳定性的。我们假设，由于单体RFP的结构完整性受到损害，生色团不能很好地免受分子氧的攻击。对结构-功能关系的计算研究将使蛋白质-生色团单体RFP变体的设计成为可能，这些RFP变体可在较长时间内不透氧。这些经过计算设计的单体RFP变体将帮助实验者制备具有更好保护和显著改善的光稳定性的生色团RFP。这项工作还将开发用于未来计算设计具有改进亮度的蛋白质-生色团组合的单体RFP的工具。拟议的研究有三个具体目标：(1)用于计算研究的各种生色团的力场的参数化；(2)研究RFP单体变体蛋白质的结构完整性和波动，以确定氧可以进入和攻击生色团的关键区域。(3)识别具有特定氨基酸的蛋白质区域，这些蛋白质区域可以暂时承载氧气，并在适当的时间将其释放，以积极帮助削弱氧气向体内的运输。确定这些区域将允许系统的计算-实验策略的氨基酸取代，以创造更多的保护性蛋白质，并修改蛋白质-发色团的相互作用，以增强系统的荧光性质。 与公共健康相关：这项研究的目标是提高一类重要的分子红色荧光蛋白的有用性。荧光蛋白是非常有价值的生物化学标志物，可以在分子水平上监测细胞生物学。荧光蛋白被用来阐明健康细胞的生化途径，并揭示导致疾病的分子问题。

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. PUBLIC HEALTH RELEVANCE: The goal of this research is to improve the usefulness of an important class of molecules called red fluorescent proteins. Fluorescent proteins are extremely valuable biochemical markers that allow the 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.