1. Ratiometric fluorescent probes for sensing interactions of peptides with their molecular targets Viktoriia Postupalenko, Andrey Klymchenko, Oleksandr Stryzhak,Vasyl Pivovarenko, Yves Mély 1. Laboratoire de Biophotonique et Pharmacologie, UMR-CNRS 7213, Faculté de Pharmacie, Université de Strasbourg, Illkirch, France 2. Department of Chemistry, Kyiv National Taras Shevchenko University, Ukraine

2. Proteins DNA/RNA Proteins Membranes  Fluorescence is universal method to report protein interactions with different targets Proteins

3. Environment-sensitive probes + _ Oil: nonpolar Poor solvation h h ’ Prodan + Water: Polar _  Environment-sensitive probes change their color with the change of polarity change of polarity

4.  Excited state proton transfer (ESIPT) results in two emission bands  Spectra highly depend on environment properties Two color probes: principles ESIPT h Normal N*Tautomeric Т* N* emission Т* emission N*Т*

5. Protein – nucleic acid interactions

6. Spectroscopic properties of the 3HC label  N*/T* band ratio strongly increases with polarity and H-donor ability  Hydration shifts the T* band position to the blue N* T* Polarity 3HC Shvadchak et al. Nucleic Acids Res. 2009

7. Peptide-nucleic acid interactions  N*/T* ratio decreases after peptide-nucleic acid interaction Peptide Proximity sensing DNA h Shvadchak et al. Nucleic Acids Res. 2009 Free peptide + SL3 RNA

8. Fluorescent amino acid analog AlaPheTrp 3HC-L-amino acid L-Tryptophane  All NC mutants preserve original peptide activity NC mutants with 3HC-amino acid

9. Interaction with nucleic acids Phe Ala Trp Free Ala peptide  Probe response correlates with 3D structures of peptide/nucleic acid complex Complex with SL3 RNA Phe Ala Trp Guzman et al. Science, 1998

10. Protein – membrane interactions

11. Spectroscopic properties of the MFL label MFL  Protic environment – one-band fluorescence  Aprotic – two emission bands + + + + + + Melittin Magainin-2 Polylysine (PLL) Model peptides:

12. Binding of the peptides to lipid membrane  Free peptide is poorly fluorescent – one emission band  Bound to liposomes – dual emission Free peptide Melittin bound to vesicles (DOPC) LUV models for the cellular membrane

13. Analysis of N-terminus insertion into membranes  = 38  = 10  Ratio of the two emission bands of the probe correlates with the depth of insertion 5.85 Å 12.15 Å 19.5 Å H 2 O,  = 80  = 2-3 8 Å 16.5 Å

14. Localization of N-terminus of peptides in the membrane 8 Å 16.5 Å + + + + + + Polylysine Melittin & Magainin-2

15. NC – vesicles interaction 0  NC interacts with negatively charged vesicles but only marginally with neutral ones  The N-terminus of peptide locates 17 Å from the center of bilayer MFL-NC

16. Conclusions  Environment-sensitive 3HC probe can be used for monitoring peptide-nucleic acids interactions.  Proposed fluorescent amino acid analog reports binding of NCp7 to oligonucleotides and site-selectively monitors the environment in NCp7 complexes.  MFL label reports binding to membrane by appearance of two emission bands and increase in quantum yield.  Ratio of the two emission bands of the probe correlates with the insertion depth (from parallax quenching).

17. Thank’s for your attention!