Computational Investigations of Monomeric Variants of Red Fluorescent Proteins

描述（由申请人提供）：本研究的目标是改善一类重要的荧光蛋白的荧光特性。荧光蛋白是分子和细胞生物学中非常有价值的生化标记物，其允许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，并揭示导致疾病的分子问题。红色荧光蛋白（RFP）对于体内应用是高度期望的，因为它们吸收并发射光谱的红色区域中的光，其中细胞自体荧光低。天然存在的RFPs是聚合的，这使得它们不适合用于标记目的。对这些RFP的结构修饰产生了单体RFP变体，但目前它们的用途是有限的，因为它们不对光稳定。我们假设，由于单体RFPs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长时间不透氧的蛋白质-发色团单体RFP变体。这些计算设计的单体RFP变体将有助于实验人员制备具有更好保护的发色团的RFP，并具有显著改善的光稳定性。这项工作还将开发工具，用于未来的计算设计单体RFPs与蛋白质-发色团组合，提高亮度。拟议的研究有三个具体的目标：（1）参数化的力场为各种生色团用于计算研究（2）调查的结构完整性和波动的蛋白质单体变体的RFPs，以确定关键区域，氧气可以进入和攻击生色团。（3）确定具有特定氨基酸的蛋白质区域，这些氨基酸可以暂时容纳氧气，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，以产生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.