Evolutions in Science Triggered by Green Fluorescent Protein (GFP) Johannes A. Schmid* and Hannah Neumeier [a] Green Fluorescent Protein (GFP) Green fluorescent protein (GFP) was discovered in the marine jellyfish Aequorea victoria as a side product after purification of aequorin, a chemiluminescent protein. Emission of blue light by aequorin leads to excitation of its companion protein GFP, thereby resulting in green fluorescence. [1] 30 years later, when the sequence of GFP was elucidated, [2] this molecule started to be developed into a valuable tool for various scientific applica- tions, as it became possible to apply cloning approaches and to use GFP either as a reporter molecule or as a fluorescent tag for fusion proteins. However, just after optimization of its fluorescence properties, which led to enhanced versions of GFP, it started to revolutionize many fields of science, especial- ly as a marker in living cells (for review, see ref. [3]). GFP is a small protein of 28 kDa with a barrel-like structure composed of 11 b sheets slightly twisted around the central axis, designated as a b-can structure [4, 5] (see Figure 1 for the similar structure of enhanced GFP (EGFP)). GFP fluorescence is caused by three cyclized and oxidized amino acids located in the center of the molecule. The process of fluorophore formation and maturation requires molecular oxygen for the generation of oxidized intermediate states of these amino acids. For this reason, GFP can only be expressed under aerobic conditions, but as soon as GFP maturation is completed, O 2 is no longer needed for fluorescence. Wild-type GFP exhibits two distinct excitation wavelengths due to the coexistence of both neutral and anionic amino acids in the chromophore. It has a major absorption maximum at 397 nm and a minor excitation peak at 475 nm. The scientific potential of a fluorescent protein was rapidly recognized after cloning of GFP. However, some of the proper- ties of wild-type GFP were not satisfactory with respect to fluo- rescence intensity, folding properties, the kinetics of fluoro- phore formation, and the biphasic excitation spectrum. There- fore, many efforts were undertaken to optimize this protein by point mutations, which finally led to the generation of a con- siderably improved GFP, with faster generation of the fluoro- phore, brighter fluorescence, correct folding at 37 8C, and a single excitation peak at 488 nm. In addition, many silent mu- tations were introduced to change the codon usage from that of the jellyfish towards the one preferred by vertebrates, in order to improve translation and expression in mammalian cells. This variant of GFP was designated as enhanced GFP (EGFP), the most commonly used GFP variant nowadays (Figure 1). Variants of GFP In parallel to improvements of the fluorescence properties of GFP, various point mutations also led to the creation of spec- tral variants of EGFP emitting blue, cyan, or yellow fluores- cence (EBFP, ECFP, and EYFP, respectively; Table1). Later on, even further ameliorations were achieved, thereby leading to the creation of Cerulean (a 2.5-fold brighter variant of ECFP [6] ) and Citrine, a variant of EYFP with lower pH and chloride sensi- tivity and better photostability and expression in organelles. [7] After development of these spectral variants of GFP, many efforts were undertaken to extend the range of fluorescence further into the red part of the spectrum. This turned out to be a difficult task, which has not been achieved by mutation of GFP so far; however it was achieved just by discovery and cloning of a red fluorescent protein from a different organism, [a] Prof. Dr. J. A. Schmid, H. Neumeier Centre for Biomolecular Medicine and Pharmacology Medical University Vienna Waehringerstrasse 13A, 1090 Vienna (Austria) Fax: (+ 43) 1-4277-9641 E-mail : johannes.schmid@meduniwien.ac.at Figure 1. The three-dimensional structure of EGFP is shown, as calculated from the coordinates of atoms derived from X-ray crystallography (protein data bank number 1S6Z [65] ). The chromophore is highlighted in yellow. The structure is depicted with Cn3D software, release 4.1, from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/entrez/). ChemBioChem 2005, 6, 1149 – 1156 DOI: 10.1002/cbic.200500029  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 1149