Investigation of BLUF photochemistry by isotopic labelling of flavin cofactor and amino acid side chains

BLUF (Blue light sensors using FAD) domains contain a so far poorly understood flavin cofactor, which is surrounded by a hydrogen bonded network, that is reversibly switched within picoseconds after a light flash. The resulting signalling state activates an associated enzyme domain or signalling pathway. Molecular details of the primary light-induced hydrogen-bond rearrangement as well as the resulting signalling processes will be analysed by vibrational spectroscopic studies. Time resolved vibrational spectra will be recorded from a picosecond- (pump-probe) to minute- (FTIR) timescale. However, interpretation of the spectra was so far precluded by the lack of band assignment. By introduction of site directed isotope labels into amino acid side chains as well as into the flavin cofactor, spectral bands will be selectively shifted and thus unambiguously assigned. This approach will lead to a detailed molecular model of BLUF photoactivation. Selectively labelled samples will be produced in E. coli, after customizing expression strains into amino acid auxotrophs. The flavin cofactor is labelled by supplementation of a riboflavin auxotrophic strain with chemically synthesized flavins, which will be imported by a genomically integrated riboflavin transporter. The combination of flavin and amino acid labelling with state-of-the-art spectroscopy will advance the research on BLUF photoreceptors and set a new basis for general protein IR spectroscopic analysis.