Structural characterization of a thiazoline-containing chromophore in an orange fluorescent protein, monomeric Kusabira Orange.
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 A 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 CNCOH group at the 2-position of the imidazolinone moiety such that the conjugated pi-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 pi-pi stacking interaction between His197 and the chromophore as well as a pi-cation interaction between Arg69 and the chromophore. These structural findings are sufficient to account for the orange emission, pH tolerance, and photostability of mKO.
Biometal Science Laboratory, RIKEN SPring-8 Center, 1-1-1, Kouto, Sayo, Hyogo 679-5148, Japan. kikuchi@spring8.or.jp
Akihiro
A
Kikuchi
Eiko
E
Fukumura
Satoshi
S
Karasawa
Hideaki
H
Mizuno
Atsushi
A
Miyawaki
Yoshitsugu
Y
Shiro
D019943:Amino Acid Substitution; D000818:Animals; D037421:Anthozoa / Q000737:chemistry / Q000235:genetics; D018360:Crystallography, X-Ray; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D055672:Static Electricity; D013844:Thiazoles / Q000737:chemistry
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D013844:Thiazoles; D049452:Green Fluorescent Proteins