Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein
Base catalysis of chromophore formation in Arg96 and Glu222 variants of green fluorescent protein
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DOI：
10.1074/jbc.m412327200
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发表时间：
2005-07-15
影响因子：
4.8
通讯作者：
Wachter, RM
中科院分区：
文献类型：
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作者：
Sniegowski, JA;Lappe, JW;Wachter, RM
在绿色荧光蛋白（GFP）中，发色团的生物合成由自发的主链缩合反应启动。Gly（67）酰胺氮与Ser（65）羰基碳的亲核加成由蛋白质折叠催化并产生杂环中间体。为了研究这一机制，我们替换了增强型GFP（EGFP）中高度保守的残基Arg（96）和Glu（222）。在R96 M变体中，发色团形成的速率大大降低（时间常数= 7.5 x 10（3）h，pH 7），并表现出pH依赖性。在E222 Q变体中，该速率在生理pH（32小时，pH 7）下也减弱，但在pH 9 - 10下加速数倍于EGFP。相反，EGFP成熟是pH无关的，并且以1小时的时间常数（pH 7 - 10）进行。R96 M和E222 Q的质谱结果表明预环化状态的积累，与限速骨架缩合一致。pH-速率曲线表明Glu（222）羧酸盐在R96 M中的表观pK（a）为6.5，Gly（67）酰胺氮在E222 Q中的表观pK（a）为9.2。这些数据表明了GFP发色团合成的模型，其中Glu（222）的羧酸盐起着一般碱的作用，促进了从Gly（67）酰胺氮或Tyr（66）α-碳的质子提取。Arg（96）通过降低各自的pKa值和稳定α-烯醇化物来实现亲电体的作用。调节质子提取基团的碱基强度可以帮助设计快速成熟的GFP，其具有用于实时监测细胞事件的改进的特征。
In green fluorescent protein (GFP), chromophore biosynthesis is initiated by a spontaneous main-chain condensation reaction. Nucleophilic addition of the Gly(67) amide nitrogen to the Ser(65) carbonyl carbon is catalyzed by the protein fold and leads to a heterocyclic intermediate. To investigate this mechanism, we substituted the highly conserved residues Arg(96) and Glu(222) in enhanced GFP (EGFP). In the R96M variant, the rate of chromophore formation is greatly reduced ( time constant = 7.5 x 10(3) h, pH 7) and exhibits pH dependence. In the E222Q variant, the rate is also attenuated at physiological pH ( 32 h, pH 7) but is accelerated severalfold beyond that of EGFP at pH 9 - 10. In contrast, EGFP maturation is pH-independent and proceeds with a time constant of 1 h ( pH 7 - 10). Mass spectrometric results for R96M and E222Q indicate accumulation of the pre-cyclization state, consistent with rate-limiting backbone condensation. The pH-rate profile implies that the Glu(222) carboxylate titrates with an apparent pK(a) of 6.5 in R96M and that the Gly(67) amide nitrogen titrates with an apparent pK(a) of 9.2 in E222Q. These data suggest a model for GFP chromophore synthesis in which the carboxylate of Glu(222) plays the role of a general base, facilitating proton abstraction from the Gly(67) amide nitrogen or the Tyr(66) alpha-carbon. Arg(96) fulfills the role of an electrophile by lowering the respective pKa values and stabilizing the alpha-enolate. Modulating the base strength of the proton-abstracting group may aid in the design of fast-maturing GFPs with improved characteristics for real-time monitoring of cellular events.