Structural Basis of X-ray-Induced Transient Photobleaching in a Photoactivatable Green Fluorescent Protein

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• 作者：Virgile Adam ; Philippe Carpentier ; Sebastien Violot ; Mickal Lelimousin ; Claudine Darnault ; G. Ulrich Nienhaus ; Dominique Bourgeois
• 刊名：Journal of the American Chemical Society
• 出版年：2009
• 出版时间：December 23, 2009
• 年：2009
• 卷：131
• 期：50
• 页码：18063-18065
• 全文大小：181K
• 年卷期：v.131,no.50(December 23, 2009)
• ISSN：1520-5126

文摘

We have observed the photoactivatable fluorescent protein IrisFP in a transient dark state with near-atomic resolution. This dark state is assigned to a radical species that either relaxes to the ground state or evolves into a permanently bleached chromophore. We took advantage of X-rays to populate the radical, which presumably forms under illumination with visible light by an electron-transfer reaction in the triplet state. The combined X-ray diffraction and in crystallo UV−vis absorption, fluorescence, and Raman data reveal that radical formation in IrisFP involves pronounced but reversible distortion of the chromophore, suggesting a transient loss of π conjugation. These results reveal that the methylene bridge of the chromophore is the Achilles’ heel of fluorescent proteins and help unravel the mechanisms of blinking and photobleaching in FPs, which are of importance in the rational design of photostable variants.