Computational Investigations of Monomeric Variants of Red Fluorescent Proteins

描述（由申请人提供）：本研究的目的是改善一类重要荧光蛋白的荧光特性。荧光蛋白是分子和细胞生物学中极有价值的生化标志物，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，揭示导致疾病的分子问题。红色荧光蛋白（rfp）在体内应用是非常理想的，因为它们吸收和发射光谱中细胞自身荧光较低的红色区域的光。自然发生的rfp是聚合的，这使得它们不适合用于标记目的。对这些RFP的结构修改产生了单体RFP变体，但目前它们的用途有限，因为它们不具有光稳定性。我们推测，由于单体rfp的结构完整性受损，发色团不能很好地保护免受分子氧的攻击。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，这些变体在很长一段时间内不被氧气渗透。这些计算设计的单体RFP变体将帮助实验人员制备具有更好保护和显著提高光稳定性的发色团的RFP。这项工作还将为未来计算设计具有提高亮度的蛋白质-发色团组合的单体rfp开发工具。提出的研究有三个具体目的：(1)参数化各种发色团的力场，用于计算研究；(2)研究rfp单体变体的结构完整性和蛋白质波动，以确定氧气可以进入和攻击发色团的关键区域。(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容纳氧气，并在适当的时候释放氧气，以积极协助氧气向内部的衰弱运输。识别这些区域将允许氨基酸取代的系统计算实验策略，以创造更多的保护性蛋白质，并修改蛋白质-发色团相互作用，以增强系统的荧光特性。

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.