Graduate School of Integrated Science and Technology, Shizuoka University, 422-8529 Shizuoka, Japan.
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, 332-0012 Saitama, Japan.
Proc Natl Acad Sci U S A. 2019 Apr 23;116(17):8301-8309. doi: 10.1073/pnas.1818836116. Epub 2019 Apr 4.
Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.
由于蓝藻光受体仅需单个紧凑结构域即可进行发色团掺入以及吸收可见光光谱（包括长波长远红区域），因此这些分子已被广泛关注并应用于生物成像和光遗传学。然而，大多数蓝藻光受体存在一个缺点，即无法在哺乳动物细胞中掺入藻红胆素。在本研究中，我们专注于胆绿素（BV），这是一种内源性哺乳动物发色团，可吸收远红区域，并发现仅替换四个残基就足以将 BV 排斥型蓝藻光受体转化为 BV 可接受的分子。我们成功地确定了其中一种工程化分子 AnPixJg2_BV4 的晶体结构，分辨率为 1.6 Å。该结构确定了不寻常的共价键连接，导致 BV 深入插入蛋白质口袋。这四个突变残基有助于减少更深插入引起的空间位阻。我们将这些残基引入到其他结构域中，其中一个结构域，NpF2164g5_BV4，在体内从哺乳动物肝脏产生了明亮的近红外荧光。总之，本研究不仅为蓝藻光受体结合 BV 提供了分子基础，而且还为将蓝藻光受体应用于深哺乳动物组织的可视化和调控开辟了合理的策略。
