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*

Protein – nucleic acid Protein – nucleic acid interactionsinteractions

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

Protein – membrane Protein – membrane interactionsinteractions

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).

Thank’s for your attention!Thank’s for your attention!