Non-fluorescent mutant of green fluorescent protein sheds light on the mechanism of chromophore formation

被引：8
作者：
Bartkiewicz, Malgorzata [1 ]
Kazazic, Sasa [2 ]
Krasowska, Joanna [1 ]
Clark, Patricia L. [3 ]
Wielgus-Kutrowska, Beata [1 ]
Bzowska, Agnieszka [1 ]
机构：
[1] Univ Warsaw, Fac Phys, Inst Expt Phys, Div Biophys, Pasteura 5, PL-02093 Warsaw, Poland
[2] Inst Ruder Boskovic, Zagreb, Croatia
[3] Univ Notre Dame, Dept Chem & Biochem, Notre Dame, IN 46556 USA
来源：
FEBS LETTERS | 2018年 / 592卷 / 09期
关键词：
chromophore maturation; green fluorescent protein; ms/ms spectrometry; S65T mutant; S65T/G67A mutant; MATURATION;
D O I：
10.1002/1873-3468.13051
中图分类号：
Q5 [生物化学]; Q7 [分子生物学];
学科分类号：
071010 ; 081704 ;
摘要：
The mechanism of green fluorescent protein (GFP) chromophore formation is still not clearly defined. Two mechanisms have been proposed: cyclisation-dehydration-oxidation (Mechanism A) and cyclisation-oxidation-dehydration (Mechanism B). To distinguish between these mechanisms, we generated a non-fluorescent mutant of GFP, S65T/G67A-GFP. This mutant folds to a stable, native-like structure but lacks fluorescence due to interruption of the chromophore maturation process. Mass spectrometric analysis of peptides derived from this mutant reveal that chromophore formation follows only mechanism A, but that the final oxidation reaction is suppressed. This result is unexpected within the pool of examined GFP mutants, since for the wild-type GFP, there is strong support for mechanism B.
引用
收藏
页码：1516 / 1523
页数：8
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