ИСТИНА

The green fluorescent protein (GFP) and its homologues have become a very popular instrument in biology as a fluorescent tag. The fluorescent properties of these proteins are provided by the chromophore, which is formed upon an autocatalytic cyclization and subsequent oxidation of three amino acid residues. It is known, that the chromophore surrounding has a great influence on the fluorescent properties of a mature protein. Understanding of its biosynthesis mechanism is important to realize the direct modification of the protein properties, e.g. to synthesise red fluorescent proteins with absorption/emission bands within optical window of biological tissues. The mechanism of this reaction is still unknown. Several schemes were proposed, but none was finally confirmed. In this work an analysis of probable pathways of the biosynthesis of the GFP (Ser65Thr, PDB ID: 1EMA) chromophore is presented. The mechanisms of the cyclization and oxidation steps of this reaction are discussed. Residues Arg96 and Glu222 have been shown to play a catalytic role: in their absence reaction barriers rise and the rate of biosynthesis slows down according to the experiment. The oxidation mechanism has been shown to include four main steps: the chromophore deprotonation, the electron transfer from the chromophore anion to the oxygen in the triplet state, the triplet-singlet intersystem crossing and formation of the product.