Figure 2
(A) Ribbon diagram of the crystal structure of C20S AppABLUF (pdb: 1yrx), showing the FRET pair (FAD, W104). (B) Emission spectra of W64F AppABLUF in the dark- (black) and light- (red) adapted states (λexc = 295 nm). (C) Chemical structures showing ionization of tyrosine to tyrosinate. (D) Emission spectrum of W64F/W104A AppABLUF (λexc = 295 nm). The narrow peak ~330 nm is the contribution of Raman scattering of water (E) pH dependent formation of tyrosinates monitored at 295 nm for the W64F/W104A AppA BLUF mutant and for L-tyrosine in solution (inset). Fitting of the sigmoidal curve with a Boltzmann equation reveals a pK = 8.0 for the two tyrosine residues present in AppABLUF and a pK = 10.5 for L-tyrosine in solution (inset).
