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Ratiometric fluorescent probes for sensing interaction s of peptides with their molecular targets. Viktoriia Postupalenko , Andrey Klymchenko, Oleksandr Stryzhak ,Vasyl Pivovarenko, Yves Mély.

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

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

slide2

Proteins

DNA/RNA

Proteins

Membranes

Proteins

  • Fluorescence is universal method to report

protein interactions with different targets

environment sensitive probes

Water:

Polar

_

+

Oil:

nonpolar

_

+

Poor solvation

Environment-sensitive probes

h

h’

Prodan

  • Environment-sensitive probes change their color with the

change of polarity

two color probes principles

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
spectroscopic properties of the 3hc label

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
peptide nucleic acid interactions

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
fluorescent amino acid analog

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
interaction with nucleic acids

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
spectroscopic properties of the mfl label

Model peptides:

MFL

Melittin

+

+

+

+

+

+

Magainin-2

Polylysine

(PLL)

Spectroscopic properties of the MFL label
  • Protic environment – one-band fluorescence
  • Aprotic – two emission bands
binding of the peptides to lipid membrane

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
analysis of n terminus insertion into membranes

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
localization of n terminus of peptides in the membrane

+

+

+

+

+

+

16.5 Å

Polylysine

8 Å

Melittin &

Magainin-2

Localization of N-terminus of peptides in the membrane
nc vesicles interaction

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
slide16

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).
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