|Budget Amount *help
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2006: ¥4,300,000 (Direct Cost: ¥4,300,000)
Fiscal Year 2005: ¥10,500,000 (Direct Cost: ¥10,500,000)
Phototropin is a blue-light sensor protein in plants, and LOV domain binds a flavin mononucleotide (FMN) as a chromophore. A photointermediate state, S390, is formed by light-induced adduct formation between FMN and a nearby cysteine, which triggers protein structural changes for kinase activation in phototropin. In this project, we aimed at revealing the molecular mechanism of light-signal transduction in LOV domains.
By means of low-temperature FTIR spectroscopy, we revealed that the reactive cysteine is protonated in the triplet-excited state of the LOV2 domain of Adiantum phytochrome3 (phy3-LOV2) as well as its unphotolyzed state. Its hydrogen-bonding interaction is strengthened in the triplet-excited state, presumably with the FMN chromophore, and such strong interaction drives adduct formation in a microsecond timescale.
Phototropin has two LOV domains called LOV1 and LOV2. Why does it have two domains? A transgenic study suggested that only LOV2 is necessary in the function of phototropin, whereas X-ray structures are surprisingly similar between LOV1 and LOV2 domains. We compared protein structural changes between the LOV1 and LOV2 domains of phy3 by means of UV-visible and FTIR spevctroscopy. We found that protein structural changes are much larger in LOV2 than in LOV1, which is consistent with their functional roles. We concluded that plants utilize a unique protein architecture (LOV domain) for different functions by regulating their protein structural changes. Regarding the protein structural changes, we previously observed temperature-dependent FTIR spectral changes in the amide-I vibrational region of peptide backbone for phy3-LOV2, suggesting the progressive structural changes in the protein moiety. Since FMN also possesses two C=O groups, we assigned C=O stretching vibrations of FMN and protein by using ^<13>C-labeling in this article. Consequently, temperature-dependent amide-I bands are unequivocally assigned by separating the chromophore bands.