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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
Fluorescence is universal method to report
protein interactions with different targets
ProteinsProteins
Environment-sensitive probesEnvironment-sensitive probes
+ _Oil:
nonpolar Poor solvation
N
O
N
O
h h’
Prodan
+Water:Polar
_
500 550 600 6500
1
2
3
4
Polarity
Wavelength, nm
Flu
ore
scence
Inte
nsi
ty
Environment-sensitive probes change their color with the Environment-sensitive probes change their color with the
change of polaritychange of polarity
Excited state proton transfer (ESIPT) results in two emission bands Spectra highly depend on environment properties
Two color probes: principlesTwo color probes: principles
O
O
O
H
N
O
O
O
H
N
ESIPT
O
O
O
H
N
h
Normal N* Tautomeric Т*
N*emission
Т*emission
O
O
O
H
N
500 600
CH3CN
CH2Cl
2
EtOAc
Toluene
Pola
rity
(nm)
N* Т*
Spectroscopic properties of theSpectroscopic properties of the 3HC 3HC labellabel
N*/T* band ratio strongly increases with polarity and H-donor ability Hydration shifts the T* band position to the blue
400 450 500 550 600 650
0.0
0.3
0.6
0.9
1.2
1.5
1.8
Flu
ores
cenc
e In
tens
ity, a
.u.
Wavelength, nm
Buffer MeOH EtOH Acetonitrile TolueneN* T*
Po
lari
ty
O
O
OHNH
O O
OH
O
3HC
Shvadchak et al. Nucleic Acids Res. 2009
Peptide-nucleic acid interactionsPeptide-nucleic acid interactions
TERQAN
C
Zn
C C
HG K E G
RAPRKKG
C
Zn
CC
HG K E G
11
37
55
KNVKO
OHO
HN
O
O
O
3HC-NC
Q M K D
KW
NF
T A R N
N*/T* ratio decreases after peptide-nucleic acid interaction
PeptideProximitysensing
DNA
h
Shvadchak et al. Nucleic Acids Res. 2009
400 450 500 550 600 650
0.0
0.2
0.4
0.6
0.8
1.0
Flu
ores
cenc
e In
tens
ity, a
.u.
Wavelength, nm
Free peptide
+ SL3 RNA
Fluorescent amino acid analogFluorescent amino acid analog
Ala Phe Trp
OHNH2
COOH
NH2O
O
O
O
H
COOH
NH2
COOHNH
3HC-L-amino acid L-Tryptophane
All NC mutants preserve original peptide activity
NC mutants with 3HC-amino acidNC mutants with 3HC-amino acid
Interaction with nucleic acidsInteraction with nucleic acids
400 450 500 550 600 6500.0
0.2
0.4
0.6
0.8
1.0
Flu
ore
scence
Inte
nsi
ty, a.u
.
Wavelength, nm
Phe
Ala
Trp
Free Alapeptide
Probe response correlates with 3D structures of
peptide/nucleic acid complex
Complex with SL3 RNA
Phe
Ala
Trp
Guzman et al. Science, 1998
Spectroscopic properties of theSpectroscopic properties of the MFL MFL labellabel
O
O
OHNH
O
N
OH
O
MFL
Protic environment – one-band fluorescence Aprotic – two emission bands
450 500 550 600 650 700
0.0
0.2
0.4
0.6
0.8
1.0
Flu
ores
cenc
e in
tens
ity, a
.u.
Wavelength, nm
Toluene 2 EtOAc 6 Acetone 21 CH
3CN 36
H2O 80
++++
+ +
Melittin
Magainin-2
Polylysine (PLL)
Model peptides:Model peptides:
Binding of the peptides to lipid Binding of the peptides to lipid membranemembrane
Free peptide is poorly fluorescent – one emission band Bound to liposomes – dual emission
450 500 550 600 650 700
0
2
4
6
8
10
Flu
ores
cenc
e In
tens
ity, a
.u.
Wavelength, nm
Free peptide
Melittin bound tovesicles (DOPC)
LUV models for the cellular membrane
Analysis of N-terminus insertion Analysis of N-terminus insertion into membranesinto membranes
450 500 550 600 650 700
0.0
0.2
0.4
0.6
0.8
1.0
Fluo
resc
ence
Inte
nsity
, a.u
.
Wavelength, nm
Magainin-2 Melittin Polylysine
DOPC:DOPS (8:2) = 38
= 10
Ratio of the two emission bands of the
probe correlates with the depth of insertion
Magainin 2 Melittin Poly-L-lysine0.4
0.5
0.6
0.7
0.8
F (w
ith q
uen
cher
) / F
o (w
ithout quen
cher
)
surface medium deep
O
OO
O
O
PO
O
ON
5.85
Å
12.1
5 Å
19.5
Å
H2O, = 80
= 2-3
8 Å
8 Å
16.5 Å
Localization of N-terminus of Localization of N-terminus of peptides in the membranepeptides in the membrane
8 8 ÅÅ
16.5 16.5 ÅÅ
++++
++
Polylysine
O
OO
O
O
POO
O
N
O
OO
O
O
POO
O
N
O
OO
O
O
POO
O
N
O
OO
O
O
POO
O
N
Melittin &Magainin-2
NC – vesicles interactionNC – vesicles interaction
450 500 550 600 650 700
0
2
4
6
8
Flu
ores
cenc
e In
tens
ity, a
.u.
Wavelength, nm
Free peptide DOPC (0) DOPS (-1) DOPG (-1)
-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
TERQAN
C
Zn
C C
HG K E G
RAPRKKG
C
Zn
C C
HK
55MQRGNFRNQRKNVK1
E GTAR
N
NF
GQ M K D
KW
O
OHO
HN
O
O
N MFL-NC
450 500 550 600 650 700
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Fluo
resc
ence
, a.u
.
Wavelength, nm
Magainin Melittin Polylysine NCp7(1-55)
ConclusionsConclusions
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).