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Non–fluorescent mutant of green fluorescent protein sheds light on the mechanism of chromophore formation
Author(s) -
Bartkiewicz Małgorzata,
Kazazić Saša,
Krasowska Joanna,
Clark Patricia L.,
WielgusKutrowska Beata,
Bzowska Agnieszka
Publication year - 2018
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.13051
Subject(s) - green fluorescent protein , chromophore , fluorescence , mutant , chemistry , biophysics , mechanism (biology) , photochemistry , biochemistry , biology , physics , philosophy , epistemology , quantum mechanics , gene
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.