Id3, inhibitor of DNA binding and cell differentiation [Elektronische Ressource] : synthesis and conformational analysis of the full-length protein and its truncated analogues / vorgelegt von Jaroslava Svobodová

Jaroslava Svobodova

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 26
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Id3, Inhibitor of DNA Binding and Cell Differentiation:
Synthesis and Conformational Analysis of the
Full-Length Protein and its Truncated Analogues


Dissertation

zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)
der Fakultät für Chemie und Pharmazie
der Universität Regensburg




vorgelegt von
Jaroslava Svobodová
aus Semily


Regensburg 2007




















Die Arbeit wurde angeleitet von: Prof. Dr. A. Buschauer

Promotionsgesuch eingereicht am: 8. März 2007

Promotionskolloquium am 20. April 2007

Prüfungsausschuß: Vorsitzender: Prof. Dr. R.Winter
1. Gutachter: Prof. Dr. A. Buschauer
2. Gutachter: Prof. Ing. I. Stibor, CSc.
3. Prüfer: Prof. Dr. B. König Die vorliegende Arbeit wurde in der Zeit von Oktober 2003 bis Oktober 2006 an der
Fakultät für Chemie und Pharmazie der Universität Regensburg in der Arbeitsgruppe Prof.
Dr. A. Buschauer unter der Leitung von Dr. C. Cabrele angefertigt.
























Dr. C. Cabrele danke ich herzlich für die Überlassung des interessanten Themas und ihre
stetige Unterstützung.
Prof. Dr. A. Buschauer danke ich für die Möglichkeit diese Arbeit an seinem Lehrstuhl
durchführen zu dürfen.





































To my parents












Table of Contents
I. Biological Role of the Id3 Protein......................................................................................................................1
I.1 Introduction .............................................................................................................................................1
I.2 Id3 Protein Expression ............................................................................................................................2
I.3 The Role of the N- and C-Terminal Regions on the Id3 Protein Function..............................................3
I.4 Id3 Protein Stability.................................................................................................................................4
I.5 Id3 Protein Localization and Phosphorylation.........................................................................................4
I.6 Id3 in the Cell Cycle Control...................................................................................................................6
I.7 Id3 and its Cellular Pathways.....................7
I.8 Id Proteins as Targets in Cancer............................................................................................................11
I.9 Conclusion and Perspectives ...............................................................................................12
I.10 Literature .............................................................................................................................................13
II. Full-Length Id3 Protein: A Target for Peptide Chemists............................................................................19
II.1 Introduction..........................................................................................................................................19 2 Solid-Phase Synthesis of the Id3 Protein .............................................................................................19
II.3 Homodimerization of the Id3 Protein...................................................................................................21
II.4 Conformation of the Id3 Protein ..........................................................................................................22
II.5 Folding Stability of the Id3 Protein......................................................................................................25
II.6 Conclusion............................27 7 Literature..............................................................................................................................................28
III. Conformational Studies of the Id3 Helix-Loop-Helix Motif......................................................................31
III.1 Introduction.........................................................................................................................................31
III.2 Solid-Phase Synthesis of the HLH Motifs of Id3, E47, E12, and MyoD............................................31
III.3 CD Spectroscopy ................................................................................................................................33
III.4 Discussion and Conclusion.................................................................................................................37
III.5 Literature.............................................................................................................................................39
IV. Conformational Fragment Analysis of the Id3 Protein..............................................................................41
IV.1 Introduction........................................................................................................................................41
IV.2 Solid-Phase Synthesis of the N- and C-Terminal Id3 Fragments and Related Analogues .................43
IV.3 CD Spectroscopy................................................................................................................................44 4 Discussion and Conclusion.................................................................................................................55
IV.5 Literature............................................................................................................................................55
V. FRET Studies of Labeled Id3 Fragments......................................................................................................59
V.1 Introduction..........................................................................................................................................59
V.2 Design and Solid-Phase Synthesis of the Fluorescently Labeled Id3 Protein Fragments ....................60
V.3 CD Spectroscopy .................................................................................................................................62
V.4 Fluorescence Spectroscopy..................................................................................................................67
V.5 Discussion and Conclusion70
V.6 Literature..............................................................................................................................................73 VI. Synthetic Studies on Id3 Peptide Thioesters as Tools for NCL .................................................................75
VI.1 Introduction........................................................................................................................................75 2 Chemical Synthesis of C-Terminal Peptide Thioesters ......................................................................78
VI.3 Synthesis of Id3 Polypeptide Thioesters.............................................................................................82
VI.4 Conclusion..........................................................................................................................................89 5 Literature.............................90
VII. Design and Synthesis of Fluorogenic MMP-2 Substrates .........................................................................95
VII.1 Introduction.......................................................................................................................................95
VII.2 Design and Synthesis of MMP-2 Substrates .....................................................................................97
VII.3 Spectroscopic Properties of Compound VII.1 ..................................................................................99
VII.4 Enzymatic Activity of MMP-2/MMP-9 in the Presence of Peptide VII.1......................................101
VII.5 Design and Synthesis of MMP-2 Substrates Containing Fibrillogenic Domains............................103
VII.6 Spectroscopic Properties of Compounds VII.2-3............................................................................104
VII.7 Enzymatic Activity of MMP-2 in the Presence of Peptides VII.2-3...............................................105
VII.8 Discussion and Conclusion .............................................................................................................106 I.9 Literature.........................................................................................................................................108
VIII. Summary ...................................................................................................................................................111
IX. Experimental Part........115