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 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.
Published Version
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