Defining the Role of Arginine 96 in Green Fluorescent Protein Fluorophore Biosynthesis
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- 作者：Timothy I. Wood ; David P. Barondeau ; Chiharu Hitomi ; Carey J. Kassmann ; John A. Tainer ; and Elizabeth D. Getzoff
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：December 13, 2005
- 年：2005
- 卷：44
- 期：49
- 页码：16211 - 16220
- 全文大小：543K
- 年卷期：v.44,no.49(December 13, 2005)
- ISSN：1520-4995
文摘
Aequoria victoria green fluorescent protein (GFP) is a revolutionary molecular biology toolbecause of its spontaneous peptide backbone cyclization and chromophore formation from residues Ser65,Tyr66, and Gly67. Here we use structure-based design, comprehensive targeted mutagenesis, and high-resolution crystallography to probe the significant functional role of conserved Arg96 (R96) in chromophorematuration. The R96M GFP variant, in which the R96M side chain is similar in volume but lacks the R96positive charge, exhibits dramatically slower chromophore maturation kinetics (from hours to months).Comparison of the precyclized conformation of the chromophore-forming residues with the mature R96Mchromophore reveals a similar Y66 conformer, contrary to the large Y66 conformational change previouslydefined in the slowly maturing R96A variant [Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer,J. A., and Getzoff, E. D. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 12111-12116]. Comprehensive R96mutagenesis and fluorescent colony screening indicate that only the R96K substitution restores wild-typematuration kinetics. Further, we show that the slowly maturing R96A variant can be complemented witha Q183R second-site mutation designed to restore the missing R96 positive charge and rapid fluorophorebiosynthesis. Moreover, comparative structural analysis of R96M, R96K, R96A/Q183R, and wild-typeGFP reveals the importance of the presence of positive charge, rather than its exact position. Together,these structural, mutational, and biochemical results establish a pivotal role for the R96 positive chargein accelerating the GFP post-translational modification, with implications for peptide backbone cyclizationin GFP, its homologues, and related biological systems.