The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp(in) conformation) or exposed to the solvent (Trp(out) conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp(in) conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

LOV domains belong to the PAS domain superfamily, which are found in a variety of sensor proteins in organism ranging from archaea to eukaryotes, and they noncovalently bind a single flavin mononucleotide as a chromophore. We report the Raman spectra of the dark state of LOV domain in FKF1 from Arabidopsis thaliana. Spectra have been also measured for the signaling state, where a cysteinyl-flavin adduct is formed upon light irradiation. Most of the observed Raman bands are assigned on the basis of normal mode calculations using a density functional theory. We also discuss implication for the analysis of the infrared spectra of LOV domains. The comprehensive assignment provides a satisfactory framework for future investigations of the photocycle mechanism in LOV domains by vibrational spectroscopy.

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. The BLUF domain is of special interest because it uses a rigid flavin rather than an isomerizable chromophore, such as a rhodopsin or phytochrome, for its light-activation process. Crystal and solution structures of several BLUF domains were recently obtained, and their overall structures are consistent. However, there is a key ambiguity regarding the position of a conserved tryptophan (Trp-104 in AppA), in that this residue was found either close to flavin (Trp in conformation) or exposed to the solvent (Trp out conformation). The location of Trp-104 is a crucial factor in understanding the photocycle mechanism of BLUF domains, because this residue has been shown to play an essential role in the activation of AppA. In this study, we demonstrated a Trp in conformation for the BLUF domain of AppA through direct observation of the vibrational spectrum of Trp-104 by ultraviolet resonance Raman spectroscopy, and also observed light-induced conformational and environmental changes in Trp-104. This study provides a structural basis for future investigations of the photocycle mechanism of BLUF proteins.

No abstract