Photophysical characterisation and photo-cycle dynamics of LOV1-His domain of phototropin from Chlamydomonas reinhardtii with roseoflavin monophosphate cofactor.
The wild-type phototropin protein phot from the green alga Chlamydomonas reinhardtii with the blue-light photoreceptor domains LOV1 and LOV2 has flavin mononucleotide (FMN) as cofactor. For the LOV1-His domain from phot of C. reinhardtii studied here, the FMN chromophore was replaced by roseoflavin monophosphate (8-dimethylamino-8-demethyl-FMN, RoFMN) during heterologous expression in a riboflavin auxotropic Escherichia coli strain. An absorption and emission spectroscopic characterisation of the cofactor exchanged-LOV1-His (RoLOV1) domain was carried out in aqueous pH 8 phosphate buffer. The fluorescence of RoLOV1 is quenched by photo-induced charge transfer at room temperature. The photo-cyclic dynamics of RoLOV1 was observed by blue-light induced hypochromic and bathochromic absorption changes which recover on a minute timescale in the dark. Photo-excited RoFMN is thought to cause reversible protein and cofactor structural changes. Prolonged intense blue-light exposure caused photo-degradation of RoFMN in RoLOV1 to fully reduced flavin and lumichrome derivatives. Photo-cycle schemes of RoLOV1 and LOV1 are presented, and the photo-degradation dynamics of RoLOV1 is discussed.
Tyagi
A
A
Fakultät für Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
Penzkofer
A
A
Mathes
T
T
Hegemann
P
P
D000042:Absorption; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D005486:Flavin Mononucleotide / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010782:Photolysis; D056949:Phototropins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012256:Riboflavin / Q000031:analogs & derivatives / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013696:Temperature
D005415:Flavins; D056949:Phototropins; C001559:7,8-dimethylalloxazine; C009191:roseoflavin; D005486:Flavin Mononucleotide; D012256:Riboflavin