ABSTRACT
The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FAD(ox)), semiquinone form (FADH(.)), and anionic hydroquinone form (FAD(red)H(-)). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FAD(ox) to the anionic semiquinone form (FAD()(-)) with a quantum efficiency of about 2% and a back recovery time of about 10s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FAD(ox) to FADH(.), FADH(.) to FAD(red)H(-), and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/chemistry , Cryptochromes/chemistry , Amino Acid Substitution , Arabidopsis Proteins/metabolism , Cryptochromes/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Fluorescence , Tetrahydrofolates/chemistry , Tetrahydrofolates/metabolismABSTRACT
The wild-type BLUF protein Slr1694 from Synechocystis sp. PCC6803 (BLUF=blue-light sensor using FAD) has flavin adenosine dinucleotide (FAD) as natural cofactor. This light sensor causes positive phototaxis of the marine cyanobacterium. In this study the FAD cofactor of the wild-type Slr1694 was replaced by roseoflavin (RoF) and the roseoflavin derivatives RoFMN and RoFAD during heterologous expression in a riboflavin auxotrophic E. coli strain. An absorption and emission spectroscopic characterization of the cofactor-exchanged-Slr1694 (RoSlr) was carried out both under dark conditions and under illuminated conditions. The behaviour of RoF embedded in RoSlr in aqueous solution at pH 8 is compared with the behaviour of RoF in aqueous solution. The fluorescence of RoF and RoSlr is quenched by photo-induced twisted intra-molecular charge transfer at room temperature with stronger effect for RoF. The fluorescence quenching is diminished at liquid nitrogen temperature. Light exposure of RoSlr causes irreversible conversion of the protein embedded roseoflavins to 8-methylamino-flavins, 8-dimethylamino-lumichrome and 8-methylamino-lumichrome.
Subject(s)
Bacterial Proteins/chemistry , Synechocystis/chemistry , Absorption , Hydrogen-Ion Concentration , Light , Photolysis , Riboflavin/analogs & derivatives , Riboflavin/chemistry , Spectrometry, Fluorescence , Spectrophotometry, UltravioletABSTRACT
The BLUF protein Slr1694 from the cyanobacterium Synechocystis sp. PCC6803 is characterized by absorption and emission spectroscopy. Slr1694 expressed from E. coli which non-covalently binds FAD, FMN, and riboflavin (called Slr1694(I)), and reconstituted Slr1694 which dominantly contains FAD (called Slr1694(II)) are investigated. The receptor conformation of Slr1694 (dark adapted form Slr1694(r)) is transformed to the putative signalling state (light adapted form Slr1694(s)) with red-shifted absorption and decreased fluorescence efficiency by blue-light excitation. In the dark at 22 degrees C, the signalling state recovers back to the initial receptor state with a time constants of about 14.2s for Slr1694(I) and 17s for Slr1694(II). Quantum yields of signalling state formation of approximately 0.63+/-0.07 for both Slr1694(I) and Slr1694(II) were determined by transient transmission measurements and intensity dependent steady-state transmission measurements. Extended blue-light excitation causes some bound flavin conversion to the hydroquinone form and some photo-degradation, both with low quantum efficiency. The flavin-hydroquinone re-oxidizes slowly back (time constant 5-9 min) to the initial flavoquinone form in the dark. A photo-cycle dynamics scheme is presented.
Subject(s)
Bacterial Proteins/chemistry , Light Signal Transduction , Light , Receptors, Cell Surface/chemistry , Spectrum Analysis , Synechocystis/chemistry , Bacterial Proteins/radiation effects , Color , Protein Conformation/radiation effects , Receptors, Cell Surface/radiation effectsABSTRACT
The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.
Subject(s)
Phosphoric Diester Hydrolases/chemistry , Photochemistry , Rhodobacter sphaeroides/chemistry , Binding Sites , Flavin Mononucleotide/chemistry , Flavin Mononucleotide/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Flavoproteins/chemistry , Flavoproteins/metabolism , Oxidation-Reduction , Phosphoric Diester Hydrolases/metabolism , Photoreceptor Cells/chemistry , Photoreceptor Cells/metabolism , Photoreceptor Cells/radiation effects , Spectrophotometry , Time FactorsABSTRACT
An absorption and emission spectroscopic characterisation of the combined wild-type LOV1-LOV2 domain string (abbreviated LOV1/2) of phot from the green alga Chlamydomonas reinhardtii is carried out at pH 8. A LOV1/2-MBP fusion protein (MBP=maltose binding protein) and LOV1/2 with a His-tag at the C-terminus (LOV1/2-His) expressed in an Escherichia coli strain are investigated. Blue-light photo-excitation generates a non-fluorescent intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm). The photo-cycle dynamics is studied by dark-state absorption and fluorescence measurement, by following the temporal absorption and emission changes under blue and violet light exposure, and by measuring the temporal absorption and fluorescence recovery after light exposure. The fluorescence quantum yield, phi(F), of the dark adapted samples is phi(F)(LOV1/2-His) approximately 0.15 and phi(F)(LOV1/2-MBP) approximately 0.17. A bi-exponential absorption recovery after light exposure with a fast (in the several 10-s range) and a slow component (in the near 10-min range) are resolved. The quantum yield of photo-adduct formation, phi(Ad), is extracted from excitation intensity dependent absorption measurements. It decreases somewhat with rising excitation intensity. The behaviour of the combined wildtype LOV1-LOV2 double domains is compared with the behaviour of the separate LOV1 and LOV2 domains.
