Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescence

Koksharov, M.I.; Ugarova, N.N.

Авторы: Koksharov M.I., Ugarova N.N.
Журнал: Photochemical and Photobiological Sciences
Том: 12
Номер: 11
Год издания: 2013
Издательство: Royal Society of Chemistry
Местоположение издательства: United Kingdom
Первая страница: 2016
Последняя страница: 2027
DOI: 10.1039/C3PP50242B
Аннотация: Bioluminescence spectra of firefly luciferases demonstrate highly pH-sensitive spectra changing the color from green to red light when pH is lowered from alkaline to acidic. This reflects a change of ratio of the green and red emitter in the bimodal spectra of bioluminescence. We show that the mutations strongly stabilizing green (Y35N) or red (H433Y) emission compensate each other leading to WT color of firefly luciferase. We further used this compensating ability of Y35N to search strong red-shifting mutations in the C-domain of firefly luciferase by random mutagenesis. The discovered mutation E457K substantially increased the contribution of the red emitter and caused 12 nm red shift of the green emitter as well. E457 is highly conservative not only in beetle luciferases but in a whole ANL superfamily of adenylating enzymes and forms conservative structural hydrogen bond with V471. Our results suggest that the removal of this hydrogen bond only mildly affects luciferase properties and that most of the effect of E457K is caused by the introduction of positive charge. E457 forms a salt bridge with R534 in most ANL enzymes including pH-insensitive luciferases which is absent in pH-sensitive firefly luciferases. The mutant A534R shows that this salt bridge is not important for pH-sensitivity but considerably improves in vivo thermostability. Although E457 is located far from the oxyluciferin-binding site, properties of the mutant E457K suggest that it affects color by influencing AMP binding.
Добавил в систему: Кокшаров Михаил Иванович

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№	Имя	Описание	Имя файла	Размер	Добавлен
1.	Иллюстрация	Electronic Supplementary material	Supplementary_c3pp50242b.pdf	3,4 МБ	24 сентября 2013 [koksharov83]
2.	Полный текст		Koksharov_PPS-2013_LINK.pdf	63,5 КБ	21 ноября 2013 [koksharov83]

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Strategy of mutual compensation of green and red mutants of firefly luciferase identifies a mutation of the highly conservative residue E457 with a strong red shift of bioluminescenceстатья

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