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Proceedings Paper

Mechanistic aspects of GFP chromophore biogenesis
Author(s): Rebekka M. Wachter
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Paper 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 crystallography 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 H2O2. The data are most consistent with a reaction mechanism that proceeds via 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 π-overlap of the five-membered ring with the side chain adduct.

Paper Details

Date Published: 22 February 2006
PDF: 8 pages
Proc. SPIE 6098, Genetically Engineered Probes for Biomedical Applications, 609803 (22 February 2006); doi: 10.1117/12.648542
Show Author Affiliations
Rebekka M. Wachter, Arizona State Univ. (United States)

Published in SPIE Proceedings Vol. 6098:
Genetically Engineered Probes for Biomedical Applications
Alexander P. Savitsky; Rebekka M. Wachter, Editor(s)

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