The structural changes that facilitate signal transduction in blue light sensors using FAD (BLUF) photoreceptors and confer the stability of the rearranged hydrogen bond network between flavin and protein in the signaling state are still poorly understood. Here, we investigate a semiconserved Trp residue in SyPixD (Slr1694) by isotope-edited vibrational spectroscopy and site-directed mutagenesis. In the signaling state, a β-sheet structure involving the backbone of W91 is formed without apparent change of environment of the W91 indole side chain. Mutation of W91, however, significantly influences the stability of the light-adapted state, suggesting that backbone rigidity rather than discrete side-chain conformations govern the stability of the light-adapted state. On the basis of computational and crystallographic models, we interpret these changes as a +1 register shift of the β2/β5 interaction with an unaffected indole side-chain conformation, rather than a +2 register shift accompanied by an indole side-chain flip that was previously proposed on the basis of X-ray structures.
Keywords: FTIR; flavin; isotope labeling; signal transduction; tryptophan.