Division of Biophysics, Faculty of Physics, Institute of Experimental Physics, University of Warsaw, Poland.
Institut Ruđer Bošković, Zagreb, Croatia.
FEBS Lett. 2018 May;592(9):1516-1523. doi: 10.1002/1873-3468.13051. Epub 2018 Apr 20.
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.
绿色荧光蛋白（GFP）生色团形成的机制仍未明确界定。目前提出了两种机制：环化-脱水-氧化（机制 A）和环化-氧化-脱水（机制 B）。为了区分这两种机制，我们生成了 GFP 的非荧光突变体 S65T/G67A-GFP。该突变体折叠成稳定的、类似天然的结构，但由于生色团成熟过程中断而缺乏荧光。对源自该突变体的肽段进行的质谱分析表明，生色团形成仅遵循机制 A，但最终的氧化反应受到抑制。在已研究的 GFP 突变体中，这一结果出乎意料，因为对于野生型 GFP，机制 B 有很强的支持。
