A Thousand Proteins of Light: 15 Years of Advances in Fluorescent Proteins George McNamara*, 1 , and Carl A. Boswell 2 1 Analytical Imaging Core, Miller School of Medicine, University of Miami, 1450 NW 10th Ave (R-134), Miami, FL 33136 2 Molecular and Cellular Biology and Arizona Cancer Center, University of Arizona,1007 E. Lowell St., Tucson, Arizona 85721 This chapter describes a database containing over 1000 wild-type and variants of fluorescent proteins derived promarily from jellyfish, corals and other cniderians. Within the database are details on fluorescent properties of the proteins, as well as specifics regarding amino acid substitutions and references for each one. Also included are data for luminescent and photoproteins. Keywords fluorescent proteins; Aequorea; GFP; FRET; 1. Introduction The cloning of the jellyfish Aequorea victoria GFP cDNAs by Prasher et al (1992) and heterologous expression by Chalfie et al (1994), has revolutionized cell biology by making it easy to make proteins, cells, tissues, and even whole organisms glow in the dark. GFP is the light emitter, following resonance energy transfer from the calcium ion sensitive photoprotein aequorin, whose cDNA was cloned by Prasher et al (1985). Around the same time, de Wet et al (1985) cloned a firefly luciferase, which itself has had a huge impact as an ATP biosensor for industrial (no ATP implies no bacterial growth), cell, and whole organism testing. Early revisions of GFP included codon optimization for use in human cells, E.coli, yeast, and multicellular organisms, improved folding mutations and blue, cyan and yellow color changes (reviewed by Tsien, 1998). Efforts to isolate red, or even orange, fluorescent protein variants of Aequorea GFP, or from other bioluminescent beasts that contained a luciferases or photoprotein, were unsuccessful. The red color barrier was finally broken by Matz et al (1999), who had the revolutionary idea to search for fluorescent proteins in non-bioluminescent anthozoa (sea anemones and corals) from a local salt water aquarium enthusiast. The same group later isolated chromoproteins (non-fluorescent but strongly colored proteins) from many other organisms, including crustacea (copepods) and mutagenized several to make new fluorescent proteins (Gurskaya et al 2001). The early history of bioluminescence and fluorescent proteins have been reviewed in books by Shimomura (2006), Pieribone and Gruber (2006) and Zimmer (2005). The intent of this report is to summarize an electronic dataset of the more than one thousand fluorescent proteins and chromoproteins we have collected from the literature that has been published in the 15 years since Prasher et al (1992). In the interest of space, the >250 references are listed online. We also briefly refer to the over 200 eukaryotic luciferases (the number of Vibrio and other bacterial luciferases are countless), over 300 phytochromes and related molecules and one fluorescent phytofluor mutant, and over 400 phycobiliproteins (phycroerythrins, allophycocyanins). We leave for others to summarize tissue autofluorophores (NAD, flavoproteins, collagen, elastin) and plant chlorophyll and its accessory molecules 1 . *Corresponding authors: George McNamara, geomcnamara@earthlink.net , gmcnamara@med.miami.edu , 305-243-8436; Carl Boswell, cboswell@email.arizona.edu , 520-954-7053. Modern Research and Educational Topics in Microscopy. A. Méndez-Vilas and J. Díaz (Eds.) ©FORMATEX 2007 _______________________________________________________________________________________________ 287