TY - GEN
T1 - Mechanistic aspects of GFP chromophore biogenesis
AU - Wachter, Rebekka
PY - 2006
Y1 - 2006
N2 - 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 rain, 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 2O2. 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 n-overlap of the five-membered ring with the side chain adduct.
AB - 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 rain, 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 2O2. 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 n-overlap of the five-membered ring with the side chain adduct.
KW - Chromophore biogenesis
KW - Green fluorescent protein
KW - Hydrogen peroxide evolution
KW - Intrinsic cofactors
KW - Tyrosine oxidation
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U2 - 10.1117/12.648542
DO - 10.1117/12.648542
M3 - Conference contribution
AN - SCOPUS:33745200541
SN - 0819461407
SN - 9780819461407
T3 - Progress in Biomedical Optics and Imaging - Proceedings of SPIE
BT - Genetically Engineered Probes for Biomedical Applications
T2 - Genetically Engineered Probes for Biomedical Applications
Y2 - 24 January 2006 through 24 January 2006
ER -