LIFETIME ANALYSIS OF FLUORESCENT PROTEIN VARIANTS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。我的实验室开发了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”非常受欢迎，因为它可以在遗传上与各种其他蛋白质融合，从而将它们标记为红色，而不会导致它们聚集、沉淀或以其他方式错误运输。然而，mRFP1确实有明显的缺点：其荧光量子产率仅为0.25，而天然四聚体的荧光量子产率约为3倍。我们试图通过各种诱变和定向进化方法来提高量子产率，但由于缺乏高通量（最好是基于facs的）量子产率测定而受挫。测量整体亮度或强度很容易（我们有一个B-D FACS DiVa），但这反映的是功能蛋白拷贝数X消光系数X量子产率的乘积。在基于mRFP1的随机文库中，对最亮的细胞进行分选通常会发现高表达的蛋白质，偶尔会发现消光系数增加的突变体，迄今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率和激发态寿命之间存在粗略的相关性，尽管我们没有太多的数据点。mRFP1的QY为0.25，tau约为1.8 ns，而其二聚体和四聚体前身的QY分别为0.68和0.79，tau分别为3.8和4.0 ns。当然，教科书预测，对于一个自然辐射寿命固定但猝灭可变的给定发色团，量子产率和实际寿命应该成正比。以上三个例子离理论的比例性并不远。如果我们能在激发态寿命值为1.8 ns的情况下对单细胞进行分选，也许我们就能对量子产率进行分选——这至少应该与蛋白质表达水平和消光系数无关。本项目的目的是评估利用相敏流式细胞术测量荧光寿命来鉴定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.