Jennifer Sniegowski - Base-catalysis of chromophore formation in Arg96 and Glu222 variant of green fluorescent protein

Version 1

      Publication Details (including relevant citation   information):

      Journal of Biological Chemistry, 280, (2005), 26248-26255.

      Abstract:

      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 alpha -carbon. Arg96 fulfills   the role of an electrophile by lowering the respective   pKa values and stabilizing the alpha -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