Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states
Photoactivation mechanism of PAmCherry based on crystal structures of the protein in the dark and fluorescent states
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DOI：
10.1073/pnas.0909204106
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发表时间：
2009-12-15
影响因子：
11.1
通讯作者：
Verkhusha, Vladislav V.
中科院分区：
文献类型：
--
作者：
Subach, Fedor V.;Malashkevich, Vladimir N.;Verkhusha, Vladislav V.
光激活荧光蛋白(PAFP)是活细胞超分辨率成像所必需的。最近，第一个红色PAFP PAmCherry1被报道，它通过提供红色超分辨率颜色来补充可光激活的GFP。PAmCherry1原本是“暗的”，但在紫外光照射后显示出红色的荧光。为了确定PAmCherry1光激活的结构基础，我们测定了它在1.50埃和1.65埃的暗荧光和红色荧光状态下的晶体结构。在黑暗的PAmChery1中发色团的非共面结构表明，在环化的Met-66-Tyr-67-Gly-68三肽中，在Tyr-67侧链上存在N-酰亚胺官能团和单个未被氧化的C-α-C-β键。含色团多肽的MS数据表明成熟时丢失了20Da，而串联MS表明Met-66中的C-α-N键被氧化了。这些数据表明，处于暗态的PAMCherry1具有生色团N-[(E)-(5-hydroxy-1H-imidazol-2-yl)methylidene]acetamide，，据我们所知，这是以前在PAFP中没有观察到的。光活化的PAmCherry1显示出非共面阴离子类DsRed发色团，但处于反式构型。基于PAmCherry1突变体的晶体分析、MS数据和生化分析，我们提出了PAmCherry1突变体的详细光活化机理。在这个机制中，激发态的PAmCherry1发色团作为氧化剂，通过类似Koble的自由基反应从Glu-215中释放出CO2分子。Glu-215脱羧基引导碳负离子的形成，导致Tyr-67C-α-C-β键的氧化。双键延伸了Tyr-67的酚环、咪唑酮和N-酰亚胺之间的圆周率共轭，产生了红色荧光生色团。
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 angstrom and 1.65 angstrom, 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 CO2 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.