Ecole Doctorale des Sciences de la Vie et de la Santé
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Description

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


Sujets

Informations

Publié par
Publié le 01 avril 2009
Nombre de lectures 44
Langue Français
Poids de l'ouvrage 15 Mo

Extrait

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: a

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