Engineering Green Fluorescent Proteins Using an Expanded Genetic Code

Part of the Reviews in Fluorescence 2008 book series (RFLU, volume 2008)

Abstract

Green fluorescent protein from Aequorea victoria (avGFP) and its mutants, produced by classical mutagenesis, have become invaluable tools for in vivo imaging of cells and tissues. The avGFP chromophore (4-(p-hydroxybenzilidene)imidazolid-5-one) is completely encoded in its amino acid sequence. It is autocatalytically formed by the posttranslational reaction between side chains of residues 65 and 67 having molecular oxygen as the only externally required component. The fluorophore of avGFP consists of residues Ser65-dehydroTyr66-Gly67 whose cyclized backbones form the imidazolinone ring. However, the unique absorbance and fluorescence properties of GFP are not an inherent property of the isolated fluorophore. Rather, it is functional only in the context of the properly folded protein. In other words, the fluorescence is not an intrinsic property of the Ser-Tyr-Gly tripeptide; this sequence can be found in a number of other proteins such as rat platelet secretory phospholipase which exhibits no fluorescence like that of the avGFPs. Not surprisingly, the Ser-Tyr-Gly tripeptide in the structure of these proteins is not cyclized, neither is the tyrosine oxidized. Available three-dimensional structures of avGFPs as well as its fluorescent homologs from Anthozoa corals and other species such as renilla, discosoma, and anemone represent a frame to understand certain structural determinants behind the various dynamic processes. These include generation of fluorescence in the mature protein, mechanisms of chromophore formation, photoconversion between different ground states, and reorganization of the protein matrix during excited states. In other words, spectral properties of GFP are extremely sensitive probes and reporters of the interactions within the chromophore and between the chromophore and its protein matrix.

Keywords

Fermentation Codon Tyrosine Selenium Acidity 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Max-Planck-Institut für BiochemieBioFuture Research Group – Molecular BiotechnologyMartinsriedGermany

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