Ecole Doctorale des Sciences de la Vie et de la Santé

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Niveau: Supérieur, Doctorat, Bac+8
UNIVERSITE DE STRASBOURG Ecole Doctorale des Sciences de la Vie et de la Santé THESE présentée pour obtenir le grade de Docteur de l'Université de Strasbourg Discipline : Sciences du Vivant Domaine : Aspects Moléculaires et Cellulaires de la Biologie par Volodymyr SHVADCHAK SONDES FLUORESCENTES A EMISSION DUALE POUR LA CARACTERISATION D'INTERACTIONS IMPLIQUANT DES PROTEINES: APPLICATION AUX PROTEINES RETROVIRALES Soutenue le 20 avril 2009 devant la commission d'examen : Dr. Eric DEPREZ Rapporteur externe Pr. Ludovic JULLIEN Rapporteur externe Dr. Sylviane MULLER Rapporteur interne Pr.Vasyl PIVOVARENKO Examinateur Pr. Yves MELY Directiur de thèse Dr. Hugues de ROCQUIGNY Co-Directeur de thèse

  • stranded dna

  • hiv proteins

  • nucleocapsid protein

  • caracterisation d'interactions impliquant des proteines

  • oligonucleotide interactions

  • viral nucleocapsid

  • resolved fluorescence

  • application aux proteines retrovirales

  • chaperone properties


Publié le : mercredi 1 avril 2009
Lecture(s) : 44
Source : scd-theses.u-strasbg.fr
Nombre de pages : 263
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UNIVERSITE DE STRASBOURG

Ecole Doctorale des Sciences de la Vie et de la Santé


THESE

présentée pour obtenir le grade de
Docteur de l’Université de Strasbourg
Discipline : Sciences du Vivant
Domaine : Aspects Moléculaires et Cellulaires de la Biologie
par
Volodymyr SHVADCHAK

SONDES FLUORESCENTES A EMISSION DUALE POUR LA
CARACTERISATION D’INTERACTIONS IMPLIQUANT DES PROTEINES:
APPLICATION AUX PROTEINES RETROVIRALES

Soutenue le 20 avril 2009 devant la commission d’examen :

Dr. Eric DEPREZ Rapporteur externe
Pr. Ludovic JULLIEN Rapporteur externe
Dr. Sylviane MULLER Rapporteur interne
Pr.Vasyl PIVOVARENKO Examinateur
Pr. Yves MELY Directiur de thèse
Dr. Hugues de ROCQUIGNY Co-Directeur de thèse

Contents
CONTENTS
CONTENTS .................................................................................................................3
ABBREVIATIONS ......................................................................................................7
1. BIBLIOGRAPHICAL REVIEW....................................................... 9
1.1. Principles of fluorescence spectroscopy and protein labeling........ 9
1.1.1. Advantages of fluorescence ..........................................................................9
1.1.2. Fluorescence principles...............................................................................10
A) Fluorescence Spectra.........................................................................................12
B) Lifetimes and quantum yields ...........................................................................12
C) Solvent relaxation..............................................................................................13
D) Excited-state reactions.......................................................................................15
1.1.3. Fluorescent labels for proteins...................................................................16
A) Intrinsic chromophores......................................................................................16
B) GFP and other Fluorescent Proteins..................................................................17
C) Labels for FRET................................................................................................19
D) Environment-sensitive (solvatochromic) dyes ..................................................24
1.1.4. Chromophore incorporation ......................................................................29
A) Direct labeling of native proteins......................................................................29
B) Indirect labeling by click chemistry reactions...................................................32
C) “Tag-labeling”...................................................................................................33
D) Synthesis of labelled peptides ...........................................................................34
1.2. 3-Hydroxychromone dyes ................................................................ 37
1.2.1. ESIPT and ESIPT dyes...............................................................................38
1.2.2. Spectroscopic properties of 3HC derivatives............................................41
1.2.3. Kinetics study of 3-hydroxyflavones..........................................................43
1.2.4. 3HC Fluorophore design: Substitution effect...........................................45
1.2.5. Protein studies by 3HC labels ....................................................................49
1.2.6. Membrane studies by 3HF probes.............................................................52
1.3. Human immunodeficiency virus type 1 (HIV-1) ........................... 55
1.3.1. Genetic organization and structure of the virus.......................................56
A) Genetic organization .........................................................................................56
B) Viral particle......................................................................................................59
1.3.2. HIV proteins ................................................................................................60
A) Envelop proteins................................................................................................60
B) Structural proteins .............................................................................................61
C) Enzymatic proteins............................................................................................63
D) Regulatory and accessory proteins....................................................................65
1.3.3. HIV-1 life cycle ............................................................................................68
A) Pre-integration phase.........................................................................................69
B) Post-integration phase .......................................................................................70
3 Contents

1.3.4. NC................................................................................................................. 72
A) Structure............................................................................................................ 72
B) Role of NC in genome protection. Non specific RNA/DNA binding .............. 73
C) Role of NC in the virus assembly. Selective RNA binding.............................. 74
D) Role of NC in Reverse Transcription. Chaperone properties ........................... 75
E) Role of NC in Integration.................................................................................. 78
1.3.5. Vpr................................................................................................................ 80
A) Structure............................................................................................................ 80
B) Role ................................................................................................................... 81
1.3.6. Anti-HIV drug targets ................................................................................ 82
1.4. Conclusions to introduction and research objectives....................86
2. Development of two-color dyes for peptide labeling ...............87
2.1. 3-Hydroxyquinolones (3HQ) ...........................................................87
2.1.1. Fluorescent properties of 3HQs. Structural Effects ................................ 88
Article (1) 2-Aryl-3-hydroxyquinolones, a new class of dyes with solvent dependent
dual emission due to excited state intramolecular proton transfer ............................. 90
Article (2) Steric control of the excited state intramolecular proton transfer in 3-
hydroxyquinolones: steady-state and time-resolved fluorescence study.................... 90
2.1.2. Effect of basicity on ESIPT in 3-hydroxyquinolones............................... 91
Article (3) Modulation of dual fluorescence in a 3-hydroxyquinolone dye by
perturbation of its intramolecular proton transfer with solvent polarity and basicity 92
Article (4) Dual-fluorescence probe of environment basicity (hydrogen bond
accepting ability) displaying no sensitivity to polarity............................................... 92
2.1.3. Effect of viscosity......................................................................................... 93
Article (5) Modulation of Excited-State Intramolecular Proton Transfer by Viscosity
in Protic Media ........................................................................................................... 94
2.1.4. Isotopic effect: Application to water detection......................................... 95
2.2. 3-Hydroxychromones for peptide labeling.....................................97
2.2.1. 2-Furyl and 2-thienyl 3-hydroxychromones............................................. 97
2.2.2. 4’-Dimethylamino-3-hydroxyflavone ........................................................ 99
2.3. Comparison of 3HQs and 3HCs....................................................101
2.4. Synthesis and properties of amino group reactive labels ...........102
3. Sensing peptide interactions by 3HC labels...........................104
3.1. Peptide synthesis and characterization.........................................104
3.1.1. Synthesis of labeled peptides.................................................................... 104
3.1.2. Fluorescence properties of the labeled peptides..................................... 105
3.2. Peptide-DNA interactions ..............................................................107


4 Contents
3.2.1. Model systems............................................................................................107
A) Monitoring oligocation-DNA interaction........................................................108
Article (6) Excited-state intramolecular proton transfer distinguishes
microenvironments in single-and double-stranded DNA....................................110
Response of model peptides on binding to DNA .....................................................111
3.2.2. Monitoring NC – oligonucleotide interactions........................................112
Article (7) Sensing peptide-oligonucleotide interactions by a two-color
fluorescence label: application to the HIV-1 nucleocapsid protein.....................116
3.3. Protein-protein interactions........................................................... 117
3.3.1. Peptide-antibody interactions ..................................................................117
3.3.2. Vpr oligomerization ..................................................................................119
3.4. Protein-membrane interactions .................................................... 121
3.4.1. Binding of NC to membranes...................................................................122
3.4.2. Binding of Vpr(52-96) to membranes......................................................124
4. CONCLUSIONS .........................................................................129
5. PERSPECTIVES ........................................................................132
6. MATERIALS AND METHODS....................................................134
6.1. Fluorophore Synthesis.................................................................... 134
6.1.1. 3-Hydroxyquinolones (3HQs)...................................................................134
6.1.2. 3-hydroxychromones (3HCs) ...................................................................134
6.2. Peptide synthesis ............................................................................. 136
6.2.1. General methods........................................................................................136
A) Synthesis..........................................................................................................136
B) Labeling...........................................................................................................136
C) Cleavage and deprotection ..............................................................................136
D) Purification......................................................................................................136
6.2.2. Vpr (52-96) .................................................................................................137
6.2.3. NC ...............................................................................................................137
6.2.4. Tat (44-61)..................................................................................................138
6.2.5. F10C, A12C and K15C .............................................................................138
6.2.6. G5................................................................................................................138
6.3. Oligonucleotides.............................................................................. 139
6.4. Lipids and liposome preparation .................................................. 139
A) Preparation of multilamellar lipids vesicles................................................140
B) Preparation of large unilamellar vesicles by extrusion ..............................140
6.5. Physical measurements .................................................................. 141
6.5.1. Absorption spectroscopy ..........................................................................141
5 Contents

6.5.2. Steady-state fluorescence spectroscopy ................................................. 141
A) Quantum yield determination.......................................................................... 141
6.5.3. Time-resolved fluorescence spectroscopy.............................................. 142
6.5.4. Dynamic light scattering........................................................................... 142
7. APPENDIX .................................................................................144
Fluorescent dyes undergoing intramolecular proton transfer with improved sensitivity
to surface charge in lipid bilayers............................................................................. 146
Probing dynamics of HIV-1 nucleocapsid protein/target hexanucleotide complexes
by 2-aminopurine...................................................................................................... 146
Targeting the Viral Nucleocapsid Protein in Anti-HIV-1 Therapy (Review).......... 146
Identification of anti NC chaperone activity using in-house chemical library
screening (Manuscript)............................................................................................. 146
8. REFERENCES...........................................................................147
6 Abbreviations
ABBREVIATIONS

3HC 3-Hydroxychromone
3HF 3-Hydroxyflavone
3HQ 3-Hydroxyquinolone
Ch Cholesterol
DLS Dynamic Light Scattering
DMF Dimethyl formamide
DMSO Dimethyl sulfoxid
DOPC Dioleoylphosphatidylcholine
DOPG Dioleoylphosphoglycerol
DOPS Dioleoylphosphatidylserine
DPPC Dipalmitoylphosphatidylcholine
ESIPT Excited State Intramolecular Proton Transfer
LUV Large Unilamellar Vesicles
MLV Multilamellar Vesicles
N* Normal form
NMP N-methylpyrrolidone
SM Sphingomyelin
SPPS Solid Phase Peptide Synthesis
T* Tautomer form
TFA Trifluoroacetic acid
THF Tetrahydrofuran
λ Position of absorbtion maximum abs
λ Excitation wavelength ex
λ Emission wavelength em
λ N* form emission maximum N*
λ T* form emission maximum T*
N*/T* Intensity ratio of the N* and T* forms

7 Introduction

8 Introduction
1. BIBLIOGRAPHICAL REVIEW
1.1. Principles of fluorescence spectroscopy and protein
labeling
1.1.1. Advantages of fluorescence
The wide applications of fluorescence techniques in biological studies are related to its
three major advantages over other investigation methods:
• Sensitivity. Detection of single molecules is possible with proper dye selection and
experimental conditions. Whereas absorbance measurements can reliably be used at
concentrations down to several tenths of micromole, fluorescence techniques can
currently be used at picomolar concentrations- and even femtomolar for up-to-date
developments.
• High speed of response. Using fluorescence, it is possible to monitor very rapid
processes, since the response is limited only by the fluorescence lifetime within the range
−8 −1010 −10 s.
• Non-destructive character Due to its non-invasive character, the sample is not
destroyed. Thus, fluorescence can be used in living cells and tissues with limited adverse
effects.

As a consequence, fluorescence appeared as a versatile tool for a large range of applications,
as for instance:
 binding of ligands to biomolecules, including in vivo
 measurement of distances within macromolecules and biological assemblies 
 study of the dynamics of proteins folding
 measurement of ion concentrations inside living cells
 study of membrane structure and function
 cell imaging
Fluorescence is a powerful tool for studying molecular interactions in analytical
chemistry, biochemistry, cell biology, physiology, photochemistry, environmental science…
9 Introduction

1.1.2. Fluorescence principles
Fluorescence belongs to the general photophysical phenomenon called luminescence,
which corresponds to the emission of light from electronically excited states of atoms and
molecules. Concerning molecules two types of luminescence can be considered depending on the
nature of their excited state. If the emission occurs from the singlet excited state, the process is
8 9 -1
called fluorescence. The emission rates of fluorescence are typically about 10 -10 s . If the
emission occurs from the triplet excited state, the process is called phosphorescence. In this case,
6 -1
transitions to the ground state are forbidden and the emission rates are slow, about 10 -10 s .

Figure 1.1. Jablonski diagram.

Fluorescence occurs in a limited number of molecules (generally polyaromatic
hydrocarbons or heterocycles) called fluorophores or fluorescent dyes. A fluorescent probe is a
fluorophore designed to localize within a specific region of a biological specimen and/or to
respond to a specific stimulus. The process responsible for fluorescence is illustrated by the
Jablonski diagram (Fig. 1.1). Three main steps are important in the fluorescence process [1].
10 Introduction
Step 1 Excitation
A photon of energy hν supplied by an external source such as a lamp or a laser is ex
absorbed by the fluorophore, creating an excited electronic singlet state (S ). This process n
distinguishes fluorescence from other types of luminescence like radioluminescence,
electroluminescence, thermoluminescence, chemiluminescence, bioluminescence,
triboluminescence and sonoluminescence in which the excited state is induced by an ionizing
radiation (X-ray, α, β, γ), electric field, heating, chemical reaction, biological process,
electrostatic forces and ultrasounds, respectively.
Step 2 Non-radiative relaxation
The fluorophore is typically excited to one of the excited vibrational states of the first
electronic singlet state (S ). The excess of vibrational energy is rapidly transferred to the solvent, 1
through collisions with the solvent molecules. This nonradiative process is called vibrational
relaxation and occurs in the sub-picosecond range. If the fluorophore is excited to the second
electronic singlet state (S ), it rapidly falls down to the S state due to internal conversion that 2 1
corresponds to a non-radiative transition between two electronic states of the same spin
multiplicity. From S , internal conversion to S is also possible but is less efficient than 1 0
conversion from S to S , due to the much larger energy gap between S and S . Therefore, 2 1 1 0
internal conversion from S to S can compete with emission of photons (fluorescence, see 1 0
below).
Intersystem crossing is another non-radiative transition that occurs between two
isoenergetic vibrational levels belonging to electronic states of different multiplicities. Thus, an
excited molecule in the lowest vibrational level of the S state can move to the isoenergetic 1
vibrational level of the T triplet state; then vibrational relaxation brings it to the lowest 1
vibrational level of T . It is important to mention that according to the selection rules of quantum 1
mechanics, the crossing between two states with different multiplicities is forbidden. However,
due to spin-orbital coupling between the orbital magnetic moment and the spin magnetic
moment, such crossing can take place.
Other processes such as collisional quenching and fluorescence resonance energy transfer
(FRET) may also depopulate S and thus, together with internal conversion and intersystem 1
crossing, compete with fluorescence.

11

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