Ratiometric fluorescent probes for sensing interaction s of peptides with their molecular targets

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 Proteins • Fluorescence is universal method to report protein interactions with different targets

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

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

5. Protein – nucleic acid interactions

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

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

8. L-Tryptophane 3HC-L-amino acid Ala Phe Trp Fluorescent amino acid analog NC mutants with 3HC-amino acid • All NC mutants preserve original peptide activity

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

10. Protein – membrane interactions

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

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

13. H2O,  = 80  = 38 19.5 Å  = 10 12.15 Å 5.85 Å  = 2-3 Analysis of N-terminus insertion into membranes 16.5 Å 8 Å 8 Å • Ratio of the two emission bands of the probe correlates with the depth of insertion

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

15. MFL-NC NC – vesicles interaction -1 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

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!