Understanding GFP chromophore biosynthesis: Controlling backbone cyclization and modifying post-translational chemistry
Understanding GFP chromophore biosynthesis: Controlling backbone cyclization and modifying post-translational chemistry
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DOI：
10.1021/bi0479205
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发表时间：
2005-02-15
期刊：
影响因子：
2.9
通讯作者：
Getzoff, ED
中科院分区：
文献类型：
--
作者：
Barondeau, DP;Kassmann, CJ;Getzoff, ED
维多利亚水母绿色荧光蛋白（GFP）经历显著的翻译后修饰以从其组分氨基酸S65、Y 66和G67产生发色团。在这里，我们描述了突变实验，在GFP设计成4-methylideneimidazole-5-one（MIO）类似的组氨酸氨裂解酶（HAL）的翻译后活性位点亲电基团的部分，这个发色团转换。GFP变体S65 A Y 66 S（GFPhal）和四个另外的相关定点突变体的晶体结构揭示了芳香族MIO部分和GFP发色团形成和MIO生物合成的机制细节。具体地，GFP支架通过以下方式促进骨架环化：（1）通过G67酰胺孤对与残基65羰基的π * 轨道的紧密邻近对准来促进亲核攻击，以及（2）通过消除前体状态中的抑制性主链氢键来去除亲核屏障。GFP R96似乎诱导结构重排，这在对齐环化的分子轨道中很重要，通过与Y 66羰基的静电相互作用有利于G67氮去质子化，并稳定还原的烯醇化中间体。我们的结构和分析还强调了野生型GFP结构的负面设计特征，这些特征通过使发色团三肽的替代构象不稳定而有利于发色团的形成。通过为理解和控制生色团产生的驱动力和蛋白质化学提供分子基础，这项研究对通过改造氨基酸来扩展遗传密码具有意义。
The Aequorea victoria green fluorescent protein (GFP) undergoes a remarkable post-translational modification to create a chromophore out of its component amino acids S65, Y66, and G67. Here, we describe mutational experiments in GFP designed to convert this chromophore into a 4-methylideneimidazole-5-one (MIO) moiety similar to the post-translational active-site electrophile of histidine ammonia lyase (HAL). Crystallographic structures of GFP variant S65A Y66S (GFPhal) and of four additional related site-directed mutants reveal an aromatic MIO moiety and mechanistic details of GFP chromophore formation and MIO biosynthesis. Specifically, the GFP scaffold promotes backbone cyclization by (1) favoring nucleophilic attack by close proximity alignment of the G67 amide lone pair with the pi* orbital of the residue 65 carbonyl and (2) removing enthalpic barriers by eliminating inhibitory main-chain hydrogen bonds in the precursor state. GFP R96 appears to induce structural rearrangements important in aligning the molecular orbitals for ring cyclization, favor G67 nitrogen deprotonation through electrostatic interactions with the Y66 carbonyl, and stabilize the reduced enolate intermediate. Our structures and analysis also highlight negative design features of the wild-type GFP architecture, which favor chromophore formation by destabilizing alternative conformations of the chromophore tripeptide. By providing a molecular basis for understanding and controlling the driving force and protein chemistry of chromophore creation, this research has implications for expansion of the genetic code through engineering of modified amino acids.