Publication Details (including relevant citation information):
Journal of Biological Chemistry, 280, (2005), 26248-26255.
In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly67 amide nitrogen to the Ser65 carbonyl carbon is catayzed by the protein fold, and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg96 and Glu222 in EGFP (enhanced GFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5×103hours, pH 7) and exhibits pH-dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 hours, pH 7), but is accelerated several-fold beyond that of EGFP at pH 9–10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of one hour (pH 7–10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu222 carboxylate titrates with an apparent pKa of 6.5 in R96M, and that the Gly67 amide nitrogen titrates with an apparent pKa of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu222 plays the role of a general base, facilitating proton abstraction from the Gly67 amide nitrogen or the Tyr66 -carbon. Arg96 fulfills the role of an electrophile by lowering the respective pKa values and stabilizing the -enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.
Address (URL): http://hwmaint.jbc.org/cgi/content/short/280/28/26248