Fluorescent Environment-Sensitive Dyes as Reporters of Biomolecular Interactions - Progress in Molecular Biology and Translational Science - page 51

dramatically the emission color of the 3HC probe. 58 Different
oligonucleotide (ODN) sequences have been tested and the obtained
spectroscopic data were correlated with the known nuclear magnetic
resonance (NMR) structure of the peptide-ODN complexes ( Fig. 2.8 ).
The results suggested that the 3HC label senses the proximity of the
peptide-labeling site (N-terminus) to the ODN bases. This approach
allowed us to determine the peptide-ODN binding parameters and
distinguish multiple binding sites in ODNs, which is rather difficult using
other fluorescence methods. Moreover, this method was found to be
more sensitive than steady-state fluorescence anisotropy, in the case of
small ODNs. Further synthesis of the analogues of 3HC-B presenting
increased brightness and solvatochromism allowed us to significantly
improve the sensitivity of this method. 61 Currently, we are developing L -
amino acids bearing 3HC fluorophores, which is an important milestone
in the probing of peptide-DNA interactions at any desired peptide site.
4.3. Protein - protein interactions
Monitoring protein-protein interactions is of key importance in the devel-
opment of peptide-based biosensors. These interactions result commonly in
changes of the environment particularly at their interface and, therefore, can
be detected by solvatochromic labels ( Fig. 2.1 ). However, classical
solvatochromic dyes (based on NBD, Prodan, 4DMP, etc.) working on
the principle of the emission band shift and/or fluorescence quenching
are not always so efficient in highly polar media. In this respect, ESIPT-
based 3HC labels are attractive alternatives, particularly for applications with
small peptides and proteins, where the polarity of the labeling site is high.
To evaluate the possibility of using our 3HC probe for sensing
peptide-peptide interactions, the high-affinity model of interaction
between the synthetic 18-amino acid peptide pTMVP and a recombinant
antibody fragment, Fab57P, was used. 62 The pTMVP peptide, which
contains the Fab57P epitope of the tobacco mosaic virus coat protein,
was functionalized with a thiol-reactive 3HC-B derivative. The dissociation
constant K d of the interaction between pTMVP and Fab57P was largely
preserved upon labeling, as evidenced by the surface plasmon resonance
technique. The ratio of the two emission bands (N*/T*) of the pTMVP
peptide labeled at its C-terminus was found to change by 40% upon inter-
action with Fab57P. These changes corresponded to a decrease in the