Biochemistry 2008, 47, 11573–11580
11573
Structural Characterization of a Thiazoline-Containing Chromophore in an Orange Fluorescent Protein, Monomeric Kusabira Orange†,‡ Akihiro Kikuchi,*,§ Eiko Fukumura,§,| Satoshi Karasawa,⊥,@ Hideaki Mizuno,⊥ Atsushi Miyawaki,⊥ and Yoshitsugu Shiro§ Biometal Science Laboratory, RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan, Department of Life Science, Graduate School of Science, Himeji Institute of Technology/UniVersity of Hyogo, 3-2-1, Kouto, Kamigori, Ako, Hyogo 678-1297, Japan, Laboratory for Cell Function and Dynamics, AdVanced Technology DeVelopment Group, Brain Science Institute, RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan, Amalgaam Company Ltd., 2-9-3 Itabashi, Itabashi-ku, Tokyo 173-0004, Japan, and Medical and Biological Laboratories Company Ltd., 3-5-10 Marunouchi, Naka-ku, Nagoya, Aichi 460-0002, Japan ReceiVed April 23, 2008; ReVised Manuscript ReceiVed August 21, 2008
ABSTRACT:
Monomeric Kusabira Orange (mKO) is a green fluorescent protein (GFP)-like protein that emits orange light at a peak of 559 nm. We analyzed its X-ray structure at 1.65 Å and found a novel three-ring chromophore that developed autocatalytically from a Cys65-Tyr66-Glu67 tripeptide in which the side chain of Cys65 formed the third 2-hydroxy-3-thiazoline ring. As a result, the chromophore contained the CdNCOH group at the 2-position of the imidazolinone moiety such that the conjugated π-electron system of the chromophore was more extended than that of GFP but less extended than that of the Discosoma sp. red fluorescent protein (DsRed). Since a sulfur atom has potent nucleophilic character, the third 3-thiazoline ring is rapidly and completely cyclized. Furthermore, our structure reveals the presence of a π-π stacking interaction between His197 and the chromophore as well as a π-cation interaction between Arg69 and the chromophore. These structural findings are sufficient to account for the orange emission, pH tolerance, and photostability of mKO. Aequorea Victoria green fluorescent protein (GFP)1 and GFP-like proteins from animals mainly within the class Anthozoa are useful tools in the fields of molecular and cellular biology. The number of available colors has greatly increased with the production of mutants and the discovery of new GFP-like proteins (1-6). Although the overall folding in the case of the GFP-like proteins is identical, color variation can occur due to the structure of the chromophore, which is autocatalytically formed from the internal tripeptide. In the case of GFP from Aequorea, this is achieved by cyclization of the protein backbone of the Ser65-Tyr66-Gly67 sequence, leading to the formation of the 4-(p-hydroxybenzylidene)imidazolinone † This work was supported by grants from the Molecular Ensemble Program at RIKEN. ‡ The atomic coordinates and structural factors have been deposited in the Protein Data Bank (entry 2ZMU for mKO at pH 9.1 and entry 2ZMW for mKO at pH 6.0). * To whom correspondence should be addressed. Phone: +81-79158-2817. Fax: +81-791-58-2818. E-mail:
[email protected]. § RIKEN SPring-8 Center. | Himeji Institute of Technology/University of Hyogo. ⊥ Brain Science Institute, RIKEN. @ Amalgaam Co. Ltd. and Medical and Biological Laboratories Co. Ltd. 1 Abbreviations: GFP, green fluorescent protein; KO, Kusabira Orange fluorescent protein; mKO, monomeric KO; FRET, fluorescence resonance energy transfer; ESI-MS, electrospray ionization mass spectrometry; TFA, tetrafluoroacetate; TOF, time-of-flight; PEG, polyethylene glycol; CHES, 2-(N-cyclohexylamino)ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; CSD, Cambridge Structural Database; rms, root-mean-square; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
FIGURE 1: Chemical structures of the chromophore: (A) GFP, (B) DsRed, (C) zFP538, (D) mOrange, and (E) mKO.
moiety (7, 8) (Figure 1A). The chromophore can also undergo further modification, as in the case of red-emitting Discosoma sp. red fluorescent protein (DsRed) in which the Gln66-Tyr67-Gly68 tripeptide autocatalytically undergoes additional oxidation at the CR-N bond of Gln66 during the maturation process, forming CdN-CdO (acylimine) at position 2 of the imidazolinone (9) (Figure 1B). The crystal structure of the yellow fluorescent protein from Zoanthus sp., zFP538, shows a three-ring chromophore derived from
10.1021/bi800727v CCC: $40.75 2008 American Chemical Society Published on Web 10/10/2008
11574
Biochemistry, Vol. 47, No. 44, 2008
Kikuchi et al.
Table 1: Comparison of Spectral Properties of Orange-Emitting Proteins
protein
absorption/ emission maximum (nm)
molar absorption coefficient (M-1 cm-1)
quantum yield
pKa
ref
mKO Arg69Lysa Glu211Alaa Glu211Glna mOrange
548/559 564/583 561/576 558/571 548/562
51600 3500 1500 900 71000
0.60
