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
Proteins
DNA/RNA
Proteins
Membranes
Proteins
 Fluorescence is universal method to report
protein interactions with different targets
Environment-sensitive probes
_
+
nonpolar
O
h
h’
N
Prodan
_
+
Polar
Poor solvation
N
O
Water:
Fluorescence Intensity
Oil:
Polarity
4
3
2
1
0
500
550
600
Wavelength, nm
650
 Environment-sensitive probes change their color with the
change of polarity
Two color probes: principles
Tautomeric Т*
Normal N*
O
O
O
O
H
O H
Т*
emission
N*
emission
h
Т*
CH3CN
Polarity
ESIPT
O
N*
N
N
CH2Cl2
EtOAc
N
N
O
O
O
O
H
Toluene
500
600 (nm)
O
O
H
 Excited state proton transfer (ESIPT) results in two emission bands
 Spectra highly depend on environment properties
Protein – nucleic acid
interactions
Spectroscopic properties of the 3HC label
Buffer
MeOH
EtOH
Acetonitrile
Toluene
1.5
1.2
0.9
Polarity
Fluorescence Intensity, a.u.
1.8
T*
N*
O
H
N
HO
0.6
O
3HC
O
OH
O
O
0.3
0.0
400
450
500
550
600
650
Wavelength, nm
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
Free peptide
O
Fluorescence Intensity, a.u.
HO
1.0
O
H
N
O
C
N
F
O
C
KNVK
11
O
G K EG H
Zn
G K EGH
C
Q
K
M
Zn
37 W
K
D
C
C
C
RAPRKKG
TERQAN
55
T
A
R
N
3HC-NC
0.8
h
0.6
0.4
Peptide
+ SL3 RNA
Proximity
sensing
0.2
0.0
400
450
500
550
600
650
DNA
Wavelength, nm
Shvadchak et al. Nucleic Acids Res. 2009
 N*/T* ratio decreases after peptide-nucleic acid interaction
Fluorescent amino acid analog
H
COOH
HO
O
NH2
H
N
COOH
O
NH2
NH2
O
COOH
O
3HC-L-amino acid
L-Tryptophane
NC mutants with 3HC-amino acid
Ala
Phe
Trp
 All NC mutants preserve original peptide activity
Interaction with nucleic acids
Complex with SL3 RNA
Fluorescence Intensity, a.u.
1.0
0.8
0.6
Ala
Free Ala
peptide
Phe
Trp
Ala
Trp
0.4
0.2
Phe
0.0
400
450
500
550
600
650
Guzman et al. Science, 1998
Wavelength, nm
 Probe response correlates with 3D structures of
peptide/nucleic acid complex
Protein – membrane
interactions
Spectroscopic properties of the MFL label
O
H
N
HO
O
O
OH
O
Fluorescence intensity, a.u.
MFL
1.0
0.8
0.6
N
Toluene
EtOAc
Acetone
CH3CN

2
6
21
36
H2O
80
Model peptides:
Melittin
Magainin-2
0.4
0.2
Polylysine
(PLL)
0.0
450
500
550
600
Wavelength, nm
650
700
 Protic environment – one-band fluorescence
 Aprotic – two emission bands
+
+
+
+
+
+
Fluorescence Intensity, a.u.
Binding of the peptides to lipid membrane
10
8
Melittin bound to
vesicles (DOPC)
6
4
LUV models for the
cellular membrane
2
Free peptide
0
450
500
550
600
650
700
Wavelength, nm
 Free peptide is poorly fluorescent – one emission band
 Bound to liposomes – dual emission
DOPC:DOPS (8:2)
 = 38
1.0
Magainin-2
Melittin
Polylysine
0.8
 = 10
0.6
0.4
F (with quencher) / Fo (without quencher)
Fluorescence Intensity, a.u.
Analysis of N-terminus insertion into
membranes
surface
medium
deep
0.8
16.5 Å
0.7
8Å
0.6
8Å
0.5
0.4
Magainin 2
Melittin
H2O,  = 80
0.2
N
O O
P
O O
O
0.0
O
O
500
550
600
650
700
 Ratio of the two emission bands of the
probe correlates with the depth of insertion
5.85 Å
Wavelength, nm
12.15 Å
450
 = 2-3
19.5 Å
O
Poly-L-lysine
Localization of N-terminus of peptides
in the membrane
N
O
O
O
N
O
P
O
O
O
O
O
P
O
O
O
P
O
N
+
O
+
+
+
+
O
O
O
O
O
O
O
N
O
O
16.5 Å
Polylysine
8Å
Melittin &
Magainin-2
O
O
P
O
O
O
O
+
O
O
O
NC – vesicles interaction
O
HO
O
H
N
O
G
C
N
F
O
C
MQRGNFRNQRKNVK
1
N
KEGH
Zn
EG
H
GK
T
Q
C
A
M
K
R
K
Zn
N
W
D
C
C TERQAN
RAPRKKG C
55
MFL-NC
Free peptide
DOPC (0)
DOPS (-1)
DOPG (-1)
8
6
4
2
0
Magainin
Melittin
Polylysine
NCp7(1-55)
1.2
1.0
Fluorescence, a.u.
Fluorescence Intensity, a.u.
-1
0.8
0.6
0.4
0.2
0
0.0
450
500
550
600
Wavelength, nm
650
700
450
500
550
600
650
700
Wavelength, nm
 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
Conclusions
 Environment-sensitive 3HC probe can be
monitoring peptide-nucleic acids interactions.
used
for
 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).
Thank’s for your attention!