Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3.
Role of Gln1029 in the photoactivation processes of the LOV2 domain in adiantum phytochrome3.
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DOI：
10.1021/bi0494727
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发表时间：
2004-06
期刊：
影响因子：
2.9
通讯作者：
D. Nozaki;T. Iwata;T. Ishikawa;T. Todo;S. Tokutomi;H. Kandori
中科院分区：
文献类型：
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作者：
D. Nozaki;T. Iwata;T. Ishikawa;T. Todo;S. Tokutomi;H. Kandori
趋光素（phot）是植物中的一种蓝光受体。该分子具有两个FMN（黄素单核苷酸）结合结构域，称为LOV（光-氧-电压）结构域，这是PAS （per- art -sim）超家族的一个子集。光- lov结构域的照明产生共价C（4a）黄素-半胱氨酸加合物，称为S390中间态。根据含有光敏色素发色团结构域的光融合蛋白Adiantum phytochrome3 （phy3）的LOV2结构域的晶体结构，Gln1029的侧链与FMN发色团交换氢键。Gln1029是未光解状态下FMN的C(4)=O基团的氢键给体，而Gln1029是S390中FMN的N(5)-H基团的氢键受体。本文采用低温FTIR光谱法测定了phy3-LOV2突变体Q1029L蛋白的光致结构变化，并与野生型进行了比较。Q1029L低温紫外可见光谱在77 ~ 295 K下仅检测到一种中间态S390，以及野生型。在Q1029L中，FMN在1710 cm（-1）处的C(4)=O拉伸位移到1723 cm(-1)，可能是由于Gln1029和FMN之间缺少氢键。在S390形成后，野生型和Q1029L的C(4)=O基团氢键减弱。这些观察结果与未光解态和S390态的x射线晶体结构完全一致。另一方面，在Q1029L中，FMN的C(4)=O拉伸和酰胺i振动与温度无关，而在野生型中，检测到高度依赖温度的FTIR光谱。室温下，Q1029L在77 ~ 150 K时的Amide-I振动与野生型相似，但在室温下则不同。这些事实表明，在野生型中，Q1029L突变蛋白缺乏黄素-半胱氨酸加合物形成后的渐进式蛋白质结构变化，这种变化最终改变了蛋白质片段中的β片和α螺旋的结构。Gln1029与FMN发色团的氢键相互作用可能在phy3-LOV2蛋白结构变化中起重要作用。
Phototropin (phot) is a blue-light receptor in plants. The molecule has two FMN (flavin mononucleotide)-binding domains named the LOV (light-oxygen-voltage) domain, that is a subset of a PAS (per-arnt-sim) superfamily. Illumination of phot-LOV domains produces a covalent C(4a) flavin-cysteinyl adduct, which is called the S390 intermediate state. According to the crystal structures of the LOV2 domain of Adiantum phytochrome3 (phy3), a fusion protein of phot containing the phytochrome chromophoric domain, in the unphotolyzed and S390 states, and the side chain of Gln1029 switches hydrogen bonds with the FMN chromophore. Gln1029 is the hydrogen-bonding donor of the C(4)=O group of FMN in the unphotolyzed state, whereas Gln1029 is the hydrogen-bonding acceptor of the N(5)-H group of FMN in S390. In this paper, we measured the light-induced structural changes in the Q1029L mutant protein of phy3-LOV2 by means of low-temperature FTIR spectroscopy, and the obtained spectra are compared with those of the wild type. Low-temperature UV-visible spectroscopy of Q1029L detected only one intermediate state, S390, at 77-295 K, as well as the wild type. The C(4)=O stretch of FMN at 1710 cm(-1) is shifted to 1723 cm(-1) in Q1029L, presumably because of the lack of hydrogen bonds between Gln1029 and FMN. Upon formation of S390, the C(4)=O group hydrogen bond is weakened in both wild type and Q1029L. These observations are fully consistent with the X-ray crystal structures of the unphotolyzed and S390 states. On the other hand, the C(4)=O stretch of FMN and amide-I vibrations are temperature-independent in Q1029L, in contrast to wild type, in which highly temperature-dependent FTIR spectra are detected. Amide-I vibrations of Q1029L at room temperature are similar to those of the wild type at 77-150 K but not at room temperature. These facts imply that the Q1029L mutant protein lacks progressive protein structural changes following flavin-cysteinyl adduct formation in the wild type, which eventually alter structures of beta sheet and alpha helix in the protein moiety. Hydrogen-bonding interaction of Gln1029 with the FMN chromophore likely plays an important role in the protein structural changes of phy3-LOV2.