Chromophore environment provides clue to "kindling fluorescent protein" riddle.
asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.
Chudakov
Dmitriy M
DM
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Science and Evrogen, Joint Stock Company, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
Feofanov
Alexei V
AV
Mudrik
Nikolay N
NN
Lukyanov
Sergey
S
Lukyanov
Konstantin A
KA
D000409:Alanine / Q000737:chemistry; D000818:Animals; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D012615:Sea Anemones / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013053:Spectrophotometry; D014443:Tyrosine / Q000737:chemistry
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D014443:Tyrosine; D012694:Serine; D000409:Alanine