基于蛋白质在黑暗和荧光状态下的晶体结构的 PAmCherry 光活化机制。,Proceedings of the National Academy of Sciences of the United States of America

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基于蛋白质在黑暗和荧光状态下的晶体结构的 PAmCherry 光活化机制。
Proceedings of the National Academy of Sciences of the United States of America ( IF 9.4 ) Pub Date : 2009 Dec 15 , DOI: 10.1073/pnas.0909204106
Fedor V Subach ₁ , Vladimir N Malashkevich , Wendy D Zencheck , Hui Xiao , Grigory S Filonov , Steven C Almo , Vladislav V Verkhusha

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活细胞的超分辨率成像需要光活化荧光蛋白 (PAFP)。最近，报道了第一个红色 PAFP，PAmCherry1，它通过提供红色超分辨率颜色来补充可光激活的 GFP。PAmCherry1 最初是“暗色”，但在紫外-紫光照射后呈现红色荧光。为了定义 PAmCherry1 光活化的结构基础，我们分别在 1.50 A 和 1.65 A 下确定了其在暗和红色荧光状态下的晶体结构。黑暗中发色团的非共面结构 PAmChery1 表明在环化 Met-66 的 Tyr-67 侧链中存在 N-酰基亚胺官能团和单个非氧化 C(alpha)-C(beta) 键-Tyr-67-Gly-68 三肽。带有发色团的肽的 MS 数据表明成熟后损失了 20 Da，而串联质谱显示 Met-66 中的 C(alpha)-N 键被氧化。这些数据表明，暗状态下的 PAmCherry1 具有发色团 N-[(E)-(5-羟基-1H-咪唑-2-基)亚甲基]乙酰胺，据我们所知，以前在 PAFP 中没有观察到。光活化的 PAmCherry1 表现出非共面的阴离子 DsRed 样发色团，但处于反式构型。基于 PAmCherry1 突变体的晶体学分析、MS 数据和生化分析，我们提出了详细的光活化机制。在这种机制中，激发态 PAmCherry1 发色团充当氧化剂，通过类似 Koble 的自由基反应从 Glu-215 中释放 CO(2) 分子。Glu-215 脱羧引导碳负离子形成，导致 Tyr-67 C(alpha)-C(beta) 键氧化。

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Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states.

Photoactivatable fluorescent proteins (PAFPs) are required for super-resolution imaging of live cells. Recently, the first red PAFP, PAmCherry1, was reported, which complements the photo-activatable GFP by providing a red super-resolution color. PAmCherry1 is originally "dark" but exhibits red fluorescence after UV-violet light irradiation. To define the structural basis of PAmCherry1 photoactivation, we determined its crystal structure in the dark and red fluorescent states at 1.50 A and 1.65 A, respectively. The non-coplanar structure of the chromophore in the dark PAmChery1 suggests the presence of an N-acylimine functionality and a single non-oxidized C(alpha)-C(beta) bond in the Tyr-67 side chain in the cyclized Met-66-Tyr-67-Gly-68 tripeptide. MS data of the chromophore-bearing peptide indicates the loss of 20 Da upon maturation, whereas tandem MS reveals the C(alpha)-N bond in Met-66 is oxidized. These data indicate that PAmCherry1 in the dark state possesses the chromophore N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide, which, to our knowledge, has not been previously observed in PAFPs. The photoactivated PAmCherry1 exhibits a non-coplanar anionic DsRed-like chromophore but in the trans configuration. Based on the crystallographic analysis, MS data, and biochemical analysis of the PAmCherry1 mutants, we propose the detailed photoactivation mechanism. In this mechanism, the excited-state PAmCherry1 chromophore acts as the oxidant to release CO(2) molecule from Glu-215 via a Koble-like radical reaction. The Glu-215 decarboxylation directs the carbanion formation resulting in the oxidation of the Tyr-67 C(alpha)-C(beta) bond. The double bond extends the pi-conjugation between the phenolic ring of Tyr-67, the imidazolone, and the N-acylimine, resulting in the red fluorescent chromophore.

更新日期：2017-01-31

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