LIFETIME ANALYSIS OF FLUORESCENT PROTEIN VARIANTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我的实验室已经开发出一种天然四聚体红色荧光珊瑚蛋白的单体版本(Campbell等人(2002)PNAS 99：7877)。这种“mRFP1”一直很受欢迎，因为它可以在基因上与多种其他蛋白质融合，将它们标记为红色，而不会导致它们聚集、沉淀或以其他方式错误运输。然而，mRFP1确实有一个明显的缺点：它的荧光量子产率仅为0.25，而天然四聚体的荧光量子产率约高3倍。我们试图通过各种诱变和定向进化方法来增加量子产率，但由于缺乏高通量(最好是基于FACS)的量子产率分析而受挫。测量整体亮度或强度非常容易(我们有B-D FACSDIVA)，但这反映了功能蛋白质拷贝数X消光系数X量子产率的乘积。在基于mRFP1的随机文库中对最亮的细胞进行排序通常会找到高表达的蛋白质，偶尔会发现消光系数增加的突变，到目前为止从未产生过量子产率的增加。但我们已经注意到量子产额和激发态寿命之间存在大致的相关性，尽管我们没有太多的数据点。二聚体和四聚体的QY分别为0.68和0.79，T_u分别为3.8和4.0 ns。当然，教科书预测，对于给定的天然辐射寿命固定但猝灭可变的生色团，量子产额和实际寿命应该成正比。上述三个例子与理论上的相称性相距不远。如果我们可以对单个细胞的激发态寿命进行排序，得到1.8 ns的值，也许我们就可以对量子产率进行排序--这至少应该与蛋白质表达水平和消光系数无关。该项目的目标是评估利用荧光寿命的相位敏感的流式细胞术测量来识别荧光蛋白明亮变体的可行性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. My lab has developed a monomeric version of a naturally tetrameric red fluorescent coral protein (Campbell et al (2002) PNAS 99: 7877). This "mRFP1" has been very popular because it can be genetically fused to a wide variety of other proteins to label them red without causing them to aggregate, precipitate, or otherwise mis-traffic. However, mRFP1 does have a significant drawback: its fluorescence quantum yield is only 0.25 whereas the natural tetramer is about 3 fold higher. Our attempts to increase the quantum yield by various mutagenic and directed evolution approaches have been frustrated by the lack of a high-throughput (preferably FACS-based) assay for quantum yield. It is very easy to measure overall brightness or intensity (we have a B-D FACS DiVa), but this reflects the product of number of functional protein copies X extinction coefficient X quantum yield. Sorting for the brightest cells in a randomized library based on mRFP1 usually finds highly expressing proteins, occasionally finds mutants with increased extinction coefficients, and has so far never produced an increase in quantum yield. But we have noticed a rough correlation between quantum yield and excited state lifetime, though we don't have many data points. mRFP1's QY of 0.25 correlates with a tau of about 1.8 ns, whereas its dimeric and tetrameric predecessors have QYs of 0.68 and 0.79 and tau's of 3.8 and 4.0 ns respectively. Of course, the textbooks predict that quantum yield and actual lifetime should be directly proportional to each other for a given chromophore of fixed natural radiative lifetime but variable quenching. The above three examples are not too far from the theoretical proportionality. If we could sort single cells for excited state lifetime for values 1.8 ns, perhaps we would be sorting for quantum yield -- this should at least be independent of protein expression level and extinction coefficient. The goal of this project is to evaluate the feasibility of identifying bright variants of fluorescent proteins using phase sensitive flow cytometry measurements of fluorescence lifetime.