ORGN 665 |
| The appeal of fluorescence microscopy is that it allows researchers to image live cells with high spatial and temporal resolution using relatively inexpensive and generally accessible equipment. The molecular prerequisite for undertaking fluorescence imaging of a specific protein in a live cell is that the protein of interest be conjugated to a fluorophore. Until as recently as the early 1990s, satisfying this prerequisite entailed expressing and purifying the recombinant protein of interest, chemically conjugating it with an amine- or thiol-reactive dye, and microinjecting into individual cells. By the mid-1990s this approach had already been made redundant by the rapid espousal of the elegant and less intrusive technique of making protein chimeras with the Aequorea jellyfish green fluorescent protein (GFP) or one of its many variants or homologues. All of the commonly used Aequorea-derived cyan FPs (CFPs), including enhanced CFP (ECFP) and Cerulean, have a tryptophan-derived chromophore that bestows them with intrinsic and undesirable spectroscopic properties. To address these limitations, we engineered a new FP known as monomeric TFP1 (mTFP1) from the naturally occurring Clavularia cyan fluorescent protein (CFP). Our new mTFP1 has a tyrosine-derived chromophore that provides a narrower and single-peaked emission spectrum, improved brightness and photostability, and single exponential lifetime decay. The primary advantage of mTFP1 is that the Foster radius (the distance of 50% energy transfer) is significantly larger and thus the FRET signal is stronger for a given interchromophore distance. To complement mTFP1, we have recently engineered a number of spectrally orthogonal colors of FPs. Using this new series of FPs we can simultaneously label and visualize 4 different subcellular structures using a standard epi-fluorescence microscope equipped with appropriate filters. We will describe the directed evolution process used to make these new colors as well as demonstrate their utility in live cell imaging experiments. |
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Asymmetric Reactions, Combinatorial Chemistry, Molecular Recognition and Self-Assembly, Proteins, Peptides, Amino Acids and Enzyme Inhibitors
8:00 PM-10:00 PM, Wednesday, March 28, 2007 Hyatt Regency Chicago -- Riverside Center, Poster
Sci-Mix
Division of Organic Chemistry |