An In-Cell Fluorogenic Tag–Probe System for Protein Dynamics Imaging Enabled by Cell-Penetrating Peptides
journal contributionposted on 17.06.2015, 00:00 by Wataru Nomura, Nami Ohashi, Atsumi Mori, Hirokazu Tamamura
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.