Hiroshi Tsutsumi , Seiichiro Abe , Tomoaki Mino
Mar 21, 2011
Citations
0
Influential Citations
9
Citations
Journal
ChemBioChem
Abstract
Fluorescent probes are valuable molecular tools in chemical biology, and various fluorescent probes for the detection of small biological components have been developed and used for fluorescence imaging in cells. Ratiometric fluorescent probes and fluorogenic probes are particularly useful because they can suppress noise associated with background emission. Tag/probe pairs for the fluorescence imaging of proteins have recently been developed, but the number of fluorogenically active tag/probe pairs is still limited. Green fluorescent protein (GFP) is a widely used biological tool for the imaging of proteins in live cells. Its fluorescence is well controlled because the fluorophore unit is located in a unique microenvironment inside a b-barrel structure. We have previously developed a new tag/probe pair with fluorogenic activity—based on the unique characteristics of GFP—by use of the leucine zipper assembly, the ZIP tag/probe. The environment surrounding the 4-nitrobenzo-2-oxa-1,3-diazole (NBD) component of the probe peptide changes drastically from a hydrophilic state to a hydrophobic state through the formation of a 3a-helical leucine zipper structure between the tag and the probe peptides, as a result of which the bright green fluorescence of the NBD dye is induced. Use of other solvatochromic fluorophores should enable us to develop fluorogenic ZIP tag/probe pairs with other fluorescence colors. Here we describe the use of 7-diethylaminocoumarin-3-carboxylic acid (DEAC) in the development of another ZIP tag/probe pair with switchable blue fluorescence. ZIP tag/probe pairs containing the DEAC dye were designed as described in our previous report (Scheme 1). In a probe ahelical peptide, a DEAC moiety was attached to the side chain of l-a-2,3-diaminopropionic acid [Dap(DEAC)]. A Dap(DEAC) residue is situated at the X-position in the probe peptide to locate the DEAC dye in the hydrophobic region of the 3a-helical leucine zipper structure. In tag antiparallel 2a-helical peptides, two Leu residues at the Z-positions in the L2 peptide are residues complementary to the Dap(DEAC) residue of the probe peptide, and these residues are replaced by alanine (A2 peptide) or glycine (G2 peptide) so that hydrophobic spaces can be formed when the tag peptides bind to the probe peptide. The fluorescence spectra of the DEAC probe peptide showed a remarkable change as the concentration of the A2 peptide was increased. The emission maximum due to the DEAC dye shifted from 482 to 470 nm as the emission intensity increased (Figure 1 B). A DEAC-b-alanine methyl ester (7) showed emission maxima at 483 nm in HEPES buffer solution and at 470 nm in MeOH (see the Supporting Information). These results clearly suggest that through the formation of the DEAC probe/A2 peptide complex, the DEAC moiety of the probe peptide is moved from a hydrophilic environment in bulk water to a hydrophobic environment inside the 3a-helical bundle structure. The fluorescence intensity of the DEAC probe peptide at 470 nm increased up to 10.5 times on addition of A2 in a typical saturation manner (Figure 2 A, Table 1).