Structural, biochemical and biophysical characterisation of human transcription factor {RBP-J_k63 [RBP-J-kappa] [Elektronische Ressource] / von Karen Henning

Karen Henning

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 33
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Biologie
der Ludwig-Maximilians-Universität München





Structural, biochemical and biophysical
characterisation of human transcription factor
RBP-J κ









von

Karen Henning

München, im April 2006

Erklärung:

Diese Dissertation wurde im Sinne der Promotionsordung von Dr. Matthias Wilmanns, an
der Außenstelle des Europäischen Laboratoriums für Molekularbiologie (EMBL) in
Hamburg betreut und von PD Dr. Bettina Kempkes vor der Fakultät vertreten.






Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig und ohne unerlaubte Hilfe angefertigt.



München, den 20. April 2006

Karen Henning








eingereicht am 28. April 2006
Erstgutachter PD Dr. Bettina Kempkes (GSF Munich)
Zweitgutachter Prof. Dr. Dirk Eick (GSF Munich)
Mündliche Prüfung 19. Oktober 2006Table of Contents


Table of Contents
Table of Contents .......................................................................................................................i
List of Abbreviations................................................................................................................iv
1 Introduction.......................................................................................................................1
1.1 RBP-J κ 1
1.2 Protein interaction partners of RBP-J κ.............................................................................3
1.2.1 Cellular interaction partners ..............................................................................3
1.2.2 Viral interaction partners.................................................................................10
1.3 Interaction of RBP-J κ with Notch1 and EBNA2: structural aspects..............................13
1.3.1 Structure of RBP-J κ.........................................................................................13
1.3.2 Structural information about Notch1...............................................................14
1.3.3 EBNA2............................................................................................................15
1.3.4 Common and distinct features of Notch and EBNA2 .....................................17
1.4 Open questions...............................................................................................................19
1.5 Scope of this thesis .........................................................................................................20
2 Materials and Methods ....................................................................................................22
2.1 Materials.........................................................................................................................22
2.1.1 Bacterial Strains...............................................................................................22
2.1.2 Cell lines..........................................................................................................22
2.1.3 Plasmids...........................................................................................................23
2.1.4 Oligonucleotides..............................................................................................24
2.1.5 Antibodies........................................................................................................28
2.1.6 Enzymes28
2.1.7 Peptides............................................................................................................29
2.1.8 Chemicals29
2.1.9 Crystallisation screens.....................................................................................31
2.1.10 Laboratory equipment......................................................................................31
2.1.11 Synchrotron radiation sources .........................................................................32
2.1.12 Software...........................................................................................................32
2.1.13 Kits, columns and other material.....................................................................33
iTable of Contents

2.2 Methods .........................................................................................................................34
2.2.1 Bacterial cell culture........................................................................................34
2.2.2 Eucaryotic cell culture.....................................................................................37
2.2.3 DNA manipulation and analysis......................................................................39
2.2.4 Methods for the analysis of proteins and DNA/protein interactions...............42
2.2.5 Expression and purification of proteins...........................................................45
2.2.6 Isothermal titration calorimetry (ITC).............................................................54
2.2.7 Small angle X-ray scattering (SAXS)63
2.2.8 Circular dichroism (CD)..................................................................................67
3 Results.............................................................................................................................71
3.1 Expression and purification of human RBP-J κ, Notch1 and EBNA2 constructs...........71
3.1.1 Expression and purification of full-length human RBP-J κ and RBP-J κ
fragments .........................................................................................................71
Ram RamANK3.1.2 Expression and purification of Notch and Notch ............................83
3.1.3 Expression and purification of EBNA2 proteins.............................................85
3.2 Biochemical and biophysical characterization of the interaction of RBP-J κ with
Ram RamANKNotch or Notch or EBNA2 .............................................................................87
3.2.1 Binary complexes of RBP-J κ with DNA and comparison of recombinant
RBP-J κ proteins expressed in E. coli and insect cells .....................................87
Ram3.2.2 Formation of ternary complexes with RBP-J κ, DNA and Notch or
RamANKNotch .....................................................................................................99
3.2.3 Contribution of the ANK domain to RBP-J κ binding ...................................103
259-435 full-length3.2.4 The affinity of EBNA2 to nRBP is two orders of magnitude
RamANKhigher than that of Notch ....................................................................117
Ram3.3 Crystallization trials of RBP-J κ bound to DNA, peptides derived from Notch or
Ram RamANKEBNA2 and Notch or Notch .........................................................................131
4 Discussion......................................................................................................................138
4.1 Importance of RBP-J κ in the context of Epstein-Barr virus infections........................138
4.2 The binding properties of recombinant RBP-J κ depend on the expression system .....139
4.3 The conserved part of RBP-J κ has the same binding properties for DNA or Notch
proteins as its full-length counterpart ...........................................................................140
4.4 The ankyrin repeats of Notch-IC do not detectably contribute to RBP-J κ binding .....140
iiTable of Contents

4.5 The minimal RBP-J κ binding site in EBNA2 (CR5 and CR6) does not account for its
strong binding affinity ..................................................................................................145
259-4354.6 Impact of the stronger binding affinity of EBNA2 compared to that of
RamANKNotch on the cellular and Epstein-Barr viral system .........................................147
4.7 Further experiments and outlook..................................................................................149
Summary................................................................................................................................151
Zusammenfassung .................................................................................................................153
References .............................................................................................................................155
Acknowledgements ...............................................................................................................166
Lebenslauf........167

iiiList of Abbreviations

List of Abbreviations
Å Ångstrøm
aa amino acids
ADAM family of proteins with disintegrin and metalloprotease function
ANK domain of hNotch1 comprising seven ankyrin repeats
APS ammonium persulfate
ATP adenosine triphosphate
bp base pair(s)
BSA bovine serum albumin
CD circular dichroism spectropolarimetry
CIR CBF-1 interacting repressor
Cp Epstein-Barr viral C-promoter
DNA desoxyribonucleic acid
dNTP 3’-desoxyribonucleoside-5’-triphosphate
dpm disintegration per minute
DTT 1,4-dithio-DL-