Structural basis for photo-induced protein cleavage and green-to-red conversion of fluorescent protein EosFP

Abstract

Genetically encoded fusion constructs derived from fluorescent proteins (FPs) can be designed to report on a multitude of events and signals in cells, tissues, and entire organs without interfering with the complex machinery of life. EosFP is a novel FP from the scleractinian coral Lobophyllia hemprichii that switches its fluorescence emission from green (516 nm) to red (581 nm) upon irradiation with approximately 400-nm light. This property enables localized tagging of proteins and thus provides a valuable tool for tracking protein movements within live cells. Here, we present the x-ray structures of the green and red forms of WT EosFP. They reveal that formation of the red chromophore is associated with cleavage of the peptide backbone, with surprisingly little change elsewhere in the structure, and provide insights into the mechanism that generates this interesting posttranslational polypeptide modification.

Fig. 1.

Molecular structure of EosFP. (A) Ribbon diagram of the EosFP tetramer. For one of the monomers, green and red colors indicate the protein fragments created by photoconversion. The atoms in the chromophore are color-coded. (B and C) The electron densities (2 F_o-Fc) of the green (B) and red (C) chromophores are contoured at 1.2 σ.(D and E) The hydrogen bonds constraining the green (D) and red (E) chromophores are represented by dashed lines. The dashed arrow depicts the interaction between the Glu-212 carboxyl and His-62 Cβ-H that is essential for cleavage of the His-62 Nα-Cα bond.

Fig. 2.

Stereo diagram of EosFP in the vicinity of the chromophore. Carbon atoms are shown in green/black and red/gray for the green and red forms, respectively. Except for residues Phe-61 and His-62, the protein structures superimpose almost perfectly, indicating surprisingly little change in the overall structure.

Fig. 3.

Reaction mechanism explaining chromophore extension and backbone cleavage. Details are discussed in the text.