Light-Induced Structural Changes of Apoprotein and Chromophore in the Sensor of Blue Light Using FAD (BLUF) Domain of AppA for a Signaling State

Abstract

AppA is a new class blue-light receptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides and retains a characteristic flavin adenine dinucleotide (FAD)-binding domain named the "sensor of blue light using FAD" (BLUF). AppA functions as an antirepressor controlling transcription of photosynthesis genes through the direct association with a transcriptional repressor PpsR in a blue-light-dependent manner [Masuda and Bauer (2002) Cell 110, 613-623]. Illumination of AppA induces a red shift in the UV-visible absorption of FAD, which results in a signaling state of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUF domain showed relatively simple features, which were mainly composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm(-)(1). We have developed an in vitro reconstitution method, by which a fully functional BLUF domain was reconstituted from free FAD and an apoprotein for the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopically labeled with (13)C and unlabeled FAD was constituted using this method, and hydrated and deuterated samples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domain were compared with those for uniformly (15)N- and (13)C-labeled or deuterated domains as well as for the unlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore and apoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes were discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.

PixE promotes dark oligomerization of the BLUF photoreceptor PixD

“…The main interfacing residues occur along a loop (Ile-83-Val-90) located between the fourth and fifth ␤-strands, the start of the fifth ␤-strand (Trp-91-Met-93), and two C-terminal helices. At the joint between this loop and the fifth ␤-strand are highly conserved residues Trp-91 and Met-93 that undergo major conformational changes in response to light as suggested by crystallographic analysis of several BLUF proteins, FTIR, and static fluorescence studies (10,11,13,24) (Fig. 5B).…”

“…Biochemical and structural studies with AppA have led to a generalized mechanism of light perception among BLUF proteins that involves light-stimulated hydrogen-bond rearrangement between the isocyclic ring of flavin and the peptide side chain (5)(6)(7)(8)(9). Accompanying the hydrogen-bond rearrangement is a conformational change of the fifth ␤-strand and a loop between the fourth and fifth ␤-strands (10)(11)(12)(13)(14)(15).…”

The Use of Chromatin Immunoprecipitation to Define PpsR Binding Activity in Rhodobacter sphaeroides 2.4.1

“…Both in vitro (4,15,32) and bioinformatic approaches (29) enabled detailed studies of PpsR repressor activity under different growth conditions to be performed. Also, gel mobility shift analyses allowed studies of in vitro binding of PpsR to the puc promoter of R. sphaeroides to be performed under oxidizing and reducing conditions in the absence of the antirepressor AppA and other cellular molecules (4,30).…”

Structural Refinement of a Key Tryptophan Residue in the BLUF Photoreceptor AppA by Ultraviolet Resonance Raman Spectroscopy

“…The resulting plasmids pTYAppA126-W64F and pTYAppA126-Q63L-W64F were used to transform Escherichia coli strain BL21(DE3) (Novagen, San Diego, CA), and the resulting strains were used to express AppA126 W64F and Q63L/W64F mutant proteins, respectively. Previously published expression plasmids for AppA126 WT (11), Q63L (22), and W104A (24) mutant proteins were used, and the recombinant proteins were expressed and purified as described previously (11).…”

“…In contrast, flavincontaining BLUF (blue-light using flavin adenine dinucleotide (FAD)) (2) proteins show a different photoactivation mechanism that is not accompanied by prominent structural changes in the chromophore (3). In BLUF proteins, the signaling state is characterized by a light-induced~10 nm red shift of the ultraviolet-visible (UV-vis) absorption spectrum (4-10) and an~16 cm À1 downshift of the C4¼O stretching mode of the flavin ring (11)(12)(13). These relatively minor spectroscopic changes are characteristic of BLUF proteins.…”