Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 GSP, Moscow V-437, Russia.
J Biol Chem. 2010 May 21;285(21):15978-84. doi: 10.1074/jbc.M109.092320. Epub 2010 Mar 9.
The acGFPL is the first-identified member of a novel, colorless and non-fluorescent group of green fluorescent protein (GFP)-like proteins. Its mutant aceGFP, with Gly replacing the invariant catalytic Glu-222, demonstrates a relatively fast maturation rate and bright green fluorescence (lambda(ex) = 480 nm, lambda(em) = 505 nm). The reverse G222E single mutation in aceGFP results in the immature, colorless variant aceGFP-G222E, which undergoes irreversible photoconversion to a green fluorescent state under UV light exposure. Here we present a high resolution crystallographic study of aceGFP and aceGFP-G222E in the immature and UV-photoconverted states. A unique and striking feature of the colorless aceGFP-G222E structure is the chromophore in the trapped intermediate state, where cyclization of the protein backbone has occurred, but Tyr-66 still stays in the native, non-oxidized form, with C(alpha) and C(beta) atoms in the sp(3) hybridization. This experimentally observed immature aceGFP-G222E structure, characterized by the non-coplanar arrangement of the imidazolone and phenolic rings, has been attributed to one of the intermediate states in the GFP chromophore biosynthesis. The UV irradiation (lambda = 250-300 nm) of aceGFP-G222E drives the chromophore maturation further to a green fluorescent state, characterized by the conventional coplanar bicyclic structure with the oxidized double Tyr-66 C(alpha)=C(beta) bond and the conjugated system of pi-electrons. Structure-based site-directed mutagenesis has revealed a critical role of the proximal Tyr-220 in the observed effects. In particular, an alternative reaction pathway via Tyr-220 rather than conventional wild type Glu-222 has been proposed for aceGFP maturation.
acGFPL 是第一个被鉴定的新型无色非荧光绿色荧光蛋白（GFP）样蛋白家族成员。它的突变体 aceGFP，用 Gly 取代了不变的催化 Glu-222，显示出相对较快的成熟速度和明亮的绿色荧光（lambda(ex) = 480nm，lambda(em) = 505nm）。aceGFP 中的反向 G222E 单点突变导致不成熟的无色变体 aceGFP-G222E，在暴露于紫外光下时，它会不可逆地光转化为绿色荧光状态。在这里，我们呈现了 aceGFP 和 aceGFP-G222E 在不成熟和 UV 光转化状态下的高分辨率晶体结构研究。无色 aceGFP-G222E 结构的一个独特而引人注目的特征是处于被捕获的中间状态的生色团，其中蛋白质骨架已经发生环化，但 Tyr-66 仍然保持在天然的非氧化形式，C(alpha)和 C(beta)原子处于 sp(3)杂化状态。这种实验观察到的不成熟的 aceGFP-G222E 结构，其特征是咪唑啉酮和酚环的非共面排列，被归因于 GFP 生色团生物合成的中间状态之一。aceGFP-G222E 的紫外光（lambda = 250-300nm）照射进一步驱动生色团成熟到绿色荧光状态，其特征是具有传统的共面双环结构，氧化的双 Tyr-66 C(alpha)=C(beta)键和共轭的 pi 电子系统。基于结构的定点突变揭示了近端 Tyr-220 在观察到的效应中的关键作用。特别是，已经提出了一种通过 Tyr-220 而不是传统野生型 Glu-222 的替代反应途径来实现 aceGFP 成熟。
