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J. Biol. Chem., Vol. 280, Issue 28, 26248-26255, July 15, 2005

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Base Catalysis of Chromophore Formation in Arg⁹⁶ and Glu²²² Variants of Green Fluorescent Protein^*

Jennifer A. Sniegowski, Jason W. Lappe, Hetal N. Patel, Holly A. Huffman, and Rebekka M. Wachter

From the Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona 85287-1604

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 catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg⁹⁶ and Glu²²² in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced (time constant = 7.5 x 10³ h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH (32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9–10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h (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 pK_a of 6.5 in R96M and that the Gly⁶⁷ amide nitrogen titrates with an apparent pK_aa 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 pK 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.

Received for publication, November 1, 2004 , and in revised form, April 27, 2005.

^* This work was supported by National Science Foundation (NSF), Grant MCB-0213091 (to R. M. W.) and an NSF Integrative Graduate Education and Research Traineeship (to J. A. S.). NSF Grant CHE-0131222 provided funds to purchase the mass spectrometer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 480-965-8188; Fax: 480-965-2747; E-mail: RWachter{at}asu.edu.

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