N-terminal truncation does not affect the location of a conserved tryptophan in the BLUF domain of AppA from Rhodobacter sphaeroides.

The flavin-binding BLUF domains are a class of blue-light receptors, and AppA is a representative of this family. Although the crystal and solution structures of several BLUF domains have already been obtained, there is a key uncertainty regarding the position of a functionally important tryptophan (Trp104 in AppA). In the first crystal structure of an N-terminally truncated BLUF domain of AppA133 (residues 17-133), Trp104 was found in close proximity to flavin (Trp(in)), whereas in a subsequent structure with an intact N-terminus AppA126 (residues 1-126), Trp104 was exposed to the solvent (Trp(out)). A recent study compared spectroscopic properties of AppA126 and AppA133 and claimed that the Trp(in) conformation is an artifact of N-terminal truncation in AppA133. In this study, we compared the flavin vibrational spectra of AppA126 and AppA133 by using near-infrared excited Raman spectroscopy. In addition, the conformations as well as the environments of Trp104 were directly monitored by ultraviolet resonance Raman spectroscopy. These studies demonstrate that the N-terminal truncation does not induce the conformational switch between Trp(in) and Trp(out).