Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms
Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms
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DOI：
10.1111/j.1742-4658.2005.04828.x
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发表时间：
2005-08-01
期刊：
影响因子：
5.4
通讯作者：
Shimizu, T
中科院分区：
文献类型：
--
作者：
Koudo, R;Kurokawa, H;Shimizu, T
神经元PAS结构域蛋白2(NPAS2)是一种与昼夜节律相关的重要转录因子。该蛋白与BMAL1形成异源二聚体，与E-box序列结合，介导昼夜节律调节的转录。NPAS2有两个PAS结构域，N-末端有血红素结合位点。在这项研究中，我们过表达了小鼠NPAS2的PAS-B结构域(残基241-416)的野生型及其突变体，然后对分离的血红素结合蛋白进行了纯化和鉴定。野生型蛋白质的光学吸收光谱表明，Fe(III)、Fe(II)和Fe(II)-CO络合物是6配位的低自旋络合物。另一方面，共振拉曼光谱表明，Fe(III)和Fe(II)配合物都含有5配位的高自旋和6配位的低自旋配合物的混合物。根据共振拉曼光谱的nu(Fe-CO)和nu(C-O)之间的负相关关系，似乎表明血红素结合的PAS-B的轴向配体是His。共产生了6个His突变体(His266Ala、His289Ala、His300Ala、His302Ala、His329Ala和His335Ala)，并比较了它们的光吸收光谱。His335Ala突变体的光谱表明，其Fe(III)络合物是5配位的高自旋络合物，而与野生型一样，其他5个His335Ala突变体的络合物是6配位的低自旋络合物。因此，我们的结果表明PAS-B中Fe(III)的轴向配体之一是His335。此外，结合动力学表明，与抹香鲸肌红蛋白相比，血红素与NPAS2的PAS-B结构域的结合相对较弱。
Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms. This protein forms a heterodimer with BMAL1, which binds to the E-box sequence to mediate circadian rhythm-regulated transcription. NPAS2 has two PAS domains with heme-binding sites in the N-terminal portion. In this study, we overexpressed wild-type and His mutants of the PAS-B domain (residues 241-416) of mouse NPAS2 and then purified and characterized the isolated heme-bound proteins. Optical absorption spectra of the wild-type protein showed that the Fe(III), Fe(II) and Fe(II)-CO complexes are 6-co-ordinated low-spin complexes. On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5-co-ordinated high-spin and 6-co-ordinated low-spin complexes. Based on inverse correlation between nu(Fe-CO) and nu(C-O) of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme-bound PAS-B is His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared. The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5-co-ordinated high-spin complex, whereas, like the wild-type, the complexes for the five other His mutants were 6-co-ordinated low-spin complexes. Thus, our results suggest that one of the axial ligands of Fe(III) in PAS-B is His335. Also, binding kinetics suggest that heme binding to the PAS-B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin.