Nomura Wataru, Ohashi Nami, Mori Atsumi, Tamamura Hirokazu
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University 2-3-10 Kandasurugadai, Chiyoda-ku, Tokyo 101-0062, Japan.
Bioconjug Chem. 2015 Jun 17;26(6):1080-5. doi: 10.1021/acs.bioconjchem.5b00131. Epub 2015 May 15.
Fluorogenic probes are useful as molecular tools in chemical biology because they can overcome noise associated with background emission. Previously, using a leucine zipper assembly, we developed a fluorogenically active ZIP tag-probe pair. A probe peptide was designed as an α-helical peptide containing 4-nitrobenzo-2-oxa-1,3-diazole, a solvatochromic fluorescent dye. Tag peptides were designed as antiparallel 2 α-helical peptides, and the tag and probe together form the 3 α-helical bundle structure of the leucine zipper. The use of the system was limited to membrane proteins or targets on the cellular surface because the probe peptide was not compatible with cell penetration. In this study, a challenge for the fluorescent imaging of proteins inside the cells was conducted by development of the ZIP tag-probe system as the second generation. To enable the cell penetration of the probe peptide, the addition of a cell penetrating peptide sequence was tested and a probe peptide with a C-terminal octa-arginine was shown to have high affinity for the tag peptide. In addition to attachment of a CPP structure, pretreatment of cells by 1-pyrenebutyrate enhanced distribution of the probe peptide into the cytosol. Observed colocalization of fluorescence of monomer Kusabira Orange and 4-nitrobenzo-2-oxa-1,3-diazole indicates our fluorogenic tag-probe system can be utilized with tagged proteins. Following stimulation by phorbol ester, the translocation of protein kinase C was tracked by the fluorescence of 4-nitrobenzo-2-oxa-1,3-diazole, suggesting the formation of the noncovalently assembled tag-probe pairing is maintained during the translocation, even when the concentration of the probe peptide is reduced to 0.1 μM. The results indicated that the dynamic change of the protein localization by chemical stimulations can be revealed by the ZIP tag-probe system. Above all, the system is simple to handle and highly compatible with virtually any protein inside the cells.
荧光探针作为化学生物学中的分子工具非常有用，因为它们可以克服与背景发射相关的噪声。此前，我们利用亮氨酸拉链组装开发了一种具有荧光活性的ZIP标签-探针系统。探针肽被设计为一种含有4-硝基苯并-2-恶唑-1,3-二唑（一种溶剂化显色荧光染料）的α-螺旋肽。标签肽被设计为反平行的双α-螺旋肽，标签和探针一起形成亮氨酸拉链的三α-螺旋束结构。由于探针肽与细胞穿透不兼容，该系统的应用仅限于膜蛋白或细胞表面的靶点。在本研究中，通过开发第二代ZIP标签-探针系统，对细胞内蛋白质的荧光成像进行了挑战。为了使探针肽能够穿透细胞，测试了添加细胞穿透肽序列，结果表明具有C端八聚精氨酸的探针肽对标签肽具有高亲和力。除了连接细胞穿透肽结构外，用1-芘丁酸预处理细胞可增强探针肽在细胞质中的分布。观察到单体库萨比拉橙和4-硝基苯并-2-恶唑-1,3-二唑荧光的共定位表明，我们的荧光标签-探针系统可用于标记蛋白。在佛波酯刺激后，通过4-硝基苯并-2-恶唑-1,3-二唑的荧光追踪蛋白激酶C的转位，这表明即使探针肽浓度降至0.1μM，在转位过程中仍保持非共价组装的标签-探针配对的形成。结果表明，ZIP标签-探针系统可以揭示化学刺激引起的蛋白质定位的动态变化。最重要的是，该系统操作简单，与细胞内几乎任何蛋白质都具有高度兼容性。
