LIFETIME ANALYSIS OF FLUORESCENT PROTEIN VARIANTS

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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.