Mechanistic aspects of GFP chromophore biogenesis

ABSTRACT We have investigated the autocatalytic mechanism of green fluorescent protein (GFP) maturation. To this end, we have used techniques such as site-directed mutagenesis, X-ray cr ystallography and in vitro kinetics, and have monitored the reaction by fluorescence, HPLC and MALDI (matrix-assisted laser desorption ionization) mass spectrometry. In summary, we find that chromophore formation, which generally occurs within 40 to 60 min, can be accelerated dramatically under some conditions. In the E222Q variant, the rate-limiting process appears to be a function of slow proton transfer steps. Other mutagenesis data indicate that chromophore biogenesis is not driven by the aromatic character of residue 66. The GFP self-modification process involves a rate-limiting oxidation reaction that results in the production of H 2O 2. The data are most consistent with a reaction mechanism that proceeds vi a cyclization-oxidation-dehydration during in vitro maturation under aerobic conditions. The ejection of water from the heterocycle that is formed from main chain protein atoms appears to depend on the degree of -overlap of the five-m embered ring with the side chain adduct. Keywords: Green fluorescent protein, chromophore biogenesis, hydrogen peroxide evolution, intrinsic cofactors, tyrosine oxidation 1. INTRODUCTION Most researchers are familiar with gr een fluorescent protein (GFP) as a fusion tag to report on cellular events ( 1). Due to its intrinsically bright color, GFP is easy to visualize and can be used to precisely localize sub-cellular events. The family of GFP-like proteins has a profound impact on the versatility of techniques available to cell biologists studying diseases ranging from cancer to developmental disabilities to genetic defects. Highly sensitive fluorescence microscopes, used in conjunction with a large variety of gene-based fluorescent probes, provide the researcher with powerful tools to resolve cellular events ( 2-4 ). Close to two hundred new publications on GFP-related research appear in the PubMed data base each month, and many biomedical app lications for GFP as a non-invasive visual reporter can be enumerated. For example, embryonic stem cells derived from GFP-transgenic mice have been implanted into normal mice and mouse models for disease states. With this technique, the fate of implanted donor cells can be monitored visually (5). The development of therapies for a large number of genetic and neurodegenerative disorders can be facilitated by monitoring disease progression non-invasively in mouse models ( 6). In this work, we report on recent advan ces in our research on the chemistry of protein post-translational modifications, especially protein self-processing reactions, reactions that lend the protein unusual biochemical or biophysical properties. As it turns out, some aspects of the GFP self-modification process are reminiscent of other kinds of protein post-translational modifications, and some aspects are quite unique to the family of GFP-like proteins. An important class of protein post-translational modifications proceeds via oxida tive tyrosine modifications, in which molecular oxygen serves as the final electron acceptor. A good ex ample is the biosynthesis of quinone-typ e cofactors. These are redox cofactors derived from the modification of tyrosine residues, such as topaquinone (TPQ) and pyrroloquinoline quinone (PQQ) ( 7). TPQ is found in the copper amine oxidases, and is a prosthetic group covalently linked to the proteins polypeptide chain