Absorption and emission spectroscopic characterisation of the LOV2-His domain of phot from Chlamydomonas reinhardtii |
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| Institution: | 1. Department of Nursing, Brigham and Women''s Hospital, Boston, MA, USA;2. Department of Medicine, Brigham and Women''s Hospital, Boston, MA, USA;3. Renal Division, Brigham and Women''s Hospital, Boston, MA, USA;4. The Nathan E. Hellman Memorial Laboratory, Renal Division, Brigham and Women''s Hospital, Boston, MA, USA;5. Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Boston, MA, USA;1. Graz University of Technology, Institute of Biochemistry, Graz, Austria;2. University of Georgia, Department of Biochemistry and Molecular Biology, Athens, GA 30602, USA;3. Conway Institute, University College Dublin, Dublin, Ireland;4. Wylstrasse 13, CH-6052 Hergiswil (Formerly Novartis AG, Basel), Switzerland;5. University of Graz, Institute of Molecular Biosciences, Graz, Austria |
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| Abstract: | The absorption and emission behaviour of flavin mononucleotide (FMN) in the wild-type light, oxygen and voltage sensitive domain LOV2 of the photoreceptor phot from the green alga Chlamydomonas reinhardtii is studied. Actually a LOV2-His protein (LOV2 domain bound at N-terminal to 15 His aminoacids via a Gly aminoacid) expressed in an Escherichia coli strain is investigated. For fresh samples stored in the dark an initial fluorescence quantum yield of ?F = 0.12 ± 0.01 and an effective fluorescence lifetime of τF = 2.4 ± 0.1 ns are determined. Blue-light photo-excitation generates an intermediate photoproduct (flavin-C(4a)-cysteinyl adduct with absorption peak at 390 nm) resulting in an intensity-dependent fluorescence quenching. In the aqueous solutions at pH 8 approximately 3.8% of the FMN molecules are not bound to the protein binding pocket, whereas 96.2% are non-covalently bound. Even at high-intensity light excitation at 428 nm a fraction of about 7% of the non-covalently bound FMN remains non-converted to an FMN-Cys adduct because of photo-induced back-relaxation of the adduct to non-covalently bound FMN. Two holo-LOV2-His conformations with different adduct recovery time constants are revealed by spectrally and temporally resolved fluorescence and absorption measurements: A fraction of about 48% forms FMN-Cys adducts with a fast recovery time constant of τAd,f = 19 ± 2 s in the dark, and the rest forms adducts with a slow recovery time constant of τF,s = 5.5 ± 1 min. Prolonged blue light irradiation of the flavin-C(4a)-cysteinyl adducts reduces their ability to recover back in the dark to non-covalently bound FMN (photo-induced permanent adduct formation). Numerical simulations of the intensity-dependent absorption depletion reveals a quantum yield of intermediate photo-adduct formation of ?Ad = 0.9 ± 0.1. Simulation of the adduct absorption dynamics gives a quantum yield of photo-induced adduct back-relaxation of ?Ad,b = 0.15 ± 0.01 and a quantum yield of photo-induced permanent adduct formation of ?Ad,p = (2.6 ± 0.5) × 10?4. |
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