Absorption and emission spectroscopic characterisation of combined wildtype LOV1–LOV2 domain of phot from Chlamydomonas reinhardtii

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, ϕF, of the dark adapted samples is ϕF(LOV1/2-His) ≈ 0.15 and ϕF(LOV1/2-MBP) ≈ 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, ϕAd, 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.