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).
Department of Chemistry and Applied Chemistry, Graduate School of Science and Engineering, Saga University, Saga 840-8502, Japan. unno@cc.saga-u.ac.jp
Masashi
M
Unno
Yuuki
Y
Tsukiji
Kensuke
K
Kubota
Shinji
S
Masuda
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D012997:Solvents / Q000737:chemistry; D013059:Spectrum Analysis, Raman; D014364:Tryptophan / Q000737:chemistry*
C116785:AppA protein, Rhodobacter sphaeroides; D001426:Bacterial Proteins; D005420:Flavoproteins; D012997:Solvents; D014364:Tryptophan