Light-Induced Structural Changes of Apoprotein and Chromophore in the Sensor of Blue Light Using FAD (BLUF) Domain of AppA for a Signaling State
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- 作者：Shinji Masuda ; Koji Hasegawa ; and Taka-aki Ono
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：February 1, 2005
- 年：2005
- 卷：44
- 期：4
- 页码：1215 - 1224
- 全文大小：415K
- 年卷期：v.44,no.4(February 1, 2005)
- ISSN：1520-4995
文摘
AppA is a new class blue-light receptor controlling photosynthesis gene expression in thepurple 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 antirepressorcontrolling transcription of photosynthesis genes through the direct association with a transcriptionalrepressor 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 signalingstate of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUFdomain showed relatively simple features, which were mainly composed of two sets of derivative-shapedsharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm-1. We have developed an in vitro reconstitutionmethod, by which a fully functional BLUF domain was reconstituted from free FAD and an apoproteinfor the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopicallylabeled with 13C and unlabeled FAD was constituted using this method, and hydrated and deuteratedsamples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domainwere compared with those for uniformly 15N- and 13C-labeled or deuterated domains as well as for theunlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore andapoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain ofAppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of anisoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relativelylarge 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 ofblue-light perception by AppA.