. We propose to develop fast-maturing GFPs (green fluorescent proteins) by directed evolution, as guided by structural and mechanistic knowledge. An often-noted severe limitation of GFP- based technology is the poor temporal resolution of the fluorescence signal. To date, the rather sluggish rate of fluorescence acquisition (half hour at best, typically one hour) has hampered the use of GFP-like proteins as genetically encodable probes for the real-time visual detection of transcriptional activity. This work is aimed at accelerating the maturation rate of GFP several-fold, to improve the available set of GFP- based fusion tags for a wide range of cellular and biotechnological applications. The proposed experiments are designed to enhance the chemical steps of GFP chromophore bio-synthesis. Results from our laboratory indicate that the overall rate of the process may depend on the strength of a base positioned close to the chromophore-forming amino acids in the tertiary protein structure. Therefore, specific sets of residues in the immediate chromophore environment will be the primary targets for mutagenesis. A restricted randomization strategy will be employed based on chemical and structural constraints, and will be followed by optimization of the whole gene. Selection for desired features will be aided by the use of an automated laser scanning system developed in our laboratories. For the first time, the rate of color acquisition will be used directly in the selection of primary libraries, and the laser technology will allow us to determine the time required to reach the end point of maturation for hundreds of thousands to millions of colonies. Relevance to public health. Though GFP is easy to detect and allows for exact localization in the cell, it is as yet of limited use in monitoring transcriptional regulation in real time. Clearly, GFPs with more rapid maturation rates would be of tremendous advantage in the immediate detection of promoter activation by highly regulated transcription factors. Such a tool would help improve our understanding of stem cell differentiation and organismal development, biological processes that are related to a broad variety of genetic and developmental disorders. In more general terms, a fast-maturing GFP would aid in the development of therapies for a very large number of disease states including neurodegenerative and cancerous disorders. ? ? ?
| Pouwels, Lauren J; Zhang, Liping; Chan, Nam H et al. (2008) Kinetic isotope effect studies on the de novo rate of chromophore formation in fast- and slow-maturing GFP variants. Biochemistry 47:10111-22 |
