Protein - protein interaction studies by chemical cross-linking and mass spectrometry [Elektronische Ressource] / von Daniela M. Schulz

Ihr Benutzername

Sujets

• Biologie

Informations

• Publié par: martin-luther-universitat_halle-wittenberg
• Publié le: 01 janvier 2007
• Nombre de lectures: 58
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Protein - Protein Interaction Studies by Chemical
Cross-Linking and Mass Spectrometry





D i s s e r t a t i o n
zur Erlangung des akademischen Grades
Dr. rer. nat.

vorgelegt der
Naturwissenschaftlichen Fakultät I
Biowissenschaften
der Martin-Luther-Universität Halle-Wittenberg


von
Daniela M. Schulz
geboren am 28. Juli 1978 in Nürnberg




Gutachter

1. Prof. Dr. Andrea Sinz

2. Prof. Dr. Milton Stubbs

3. Prof. Dr. Michael Glocker


Eingereicht am: 14.02.2007
Tag der öffentlichen Verteidigung: 24.07.2007

urn:nbn:de:gbv:3-000012363
[ http://nbn-resolving.de/urn/resolver.pl?urn=nbn%3Ade%3Agbv%3A3-000012363 ]










































Für Oma und Opa
Content
Content....................................................................................................................... I
Bibliographische Beschreibung............................................................................. VI
Index of Tables and Figures .................................................................................. VII
Abbreviations........................................................................................................... IX
Zusammenfassung................................................................................................... 1
Summary ................................................................................................................... 5
1 Theoretical Background ...................................................................................... 9
1.1 Structural Characterization of Proteins ........................................................................9
1.1.1 Analytical Methods for Protein Three-Dimensional Structure Analysis.............................. 10
1.1.1.1 High-Resolution Techniques....................................................................................... 10
1.1.1.2 Mass Spectrometry – Based Approaches................................................................... 10
1.1.1.3 Further Methods.......................................................................................................... 11
1.1.2 Computational Protein-Protein Docking ............................................................................. 12
1.2 Mass Spectrometry.......................................................................................................13
1.2.1 Ionization Methods ............................................................................................................. 13
1.2.1.1 Matrix-Assisted Laser Desorption / Ionization (MALDI) .............................................. 13
1.2.1.2 Electrospray Ionization (ESI)....................................................................................... 13
1.2.2 Mass Analyzers .................................................................................................................. 14
1.2.2.1 Time-of-Flight Mass Spectrometry.............................................................................. 14
1.2.2.2 Fourier Transform Ion Cyclotron Resonance Mass Spectrometry.............................. 15
1.2.3 MS Fragmentation Techniques .......................................................................................... 15
1.3 Chemical Cross-Linking...............................................................................................16
1.3.1 Bifunctional Cross-Linking Reagents ................................................................................. 17
1.3.1.1 Amine-Reactive Cross-Linkers.................................................................................... 17
1.3.1.2 Sulfhydryl-Reactive Cross-Linkers.............................................................................. 18
1.3.1.3 Photo-Reactive Cross-Linkers .................................................................................... 19
1.3.1.4 Isotope-Labeled Cross-Linkers ................................................................................... 20
1.3.2 Trifunctional Cross-Linkers................................................................................................. 21
1.3.3 Zero-Length Cross-LInkers 22
1.3.4 Protein Labeling Reagents 23
1.4 Analytical Strategy........................................................................................................23
I
1.5 Studied Protein Complexes .........................................................................................24
1.5.1 Calcium-Binding Proteins ................................................................................................... 25
1.5.1.1 EF-Hand Proteins........................................................................................................ 25
1.5.1.2 Endonexin-fold ............................................................................................................ 26
1.5.2 Calmodulin / Melittin Complex............................................................................................ 26
1.5.2.1 Calmodulin .................................................................................................................. 26
1.5.2.2 Melittin......................................................................................................................... 27
1.5.2.3 Calmodulin / Target Peptide Complexes .................................................................... 27
1.5.3 Annexin A2 / p11 Complex................................................................................................. 29
1.5.3.1 Annexins...................................................................................................................... 29
1.5.3.2 Annexin A2.................................................................................................................. 29
1.5.3.3 S100 Proteins.............................................................................................................. 30
1.5.3.4 P11 (S100A10)............................................................................................................ 31
1.5.3.5 Annexin A2 / P11 Heterotetramer (A2t) ...................................................................... 31
2 Structure Determination of the Calmodulin / Melittin Complex...................... 32
2.1 Characterization of CaM and Melittin..........................................................................32
2.2 Cross-Linking Reactions..............................................................................................33
2.2.1 Gel Electrophoretic Separation of Cross-Linking Reaction Mixtures ................................. 33
2.2.2 Linear MALDI-TOFMS of Intact Cross-Linked Calmodulin / Melittin Complex.................. 35
2.2.3 In-Gel Enzymatic Digestion ................................................................................................ 37
2.3 Analysis of Cross-Linked Products ............................................................................37
2.3.1 Peptide Mass Mapping....................................................................................................... 38
2.3.2 Cross-Linked Products Between Calmodulin and Melittin ................................................. 39
2.3.2.1 Cross-Linked Products Obtained with EDC / sNHS ................................................... 39
2.3.2.2 Cross-Linked Products Obtained with sDST............................................................... 42
32.3.2.3 Cross-Linked Products Obtained with BS ................................................................. 43
2.4 Structural Model of the CaM / Melittin Complex ........................................................44
2.5 Discussion.....................................................................................................................47
2.5.1 Chemical Cross-Linking and Mass Spectrometry for Studying Mechanisms of Protein-
Protein Interactions............................................................................................................. 47
2.5.2 Structures of CaM / Peptide Complexes ............................................................................ 47
2.5.3 Structure of the CaM / Melittin Complex............................................................................. 48
3 Purification and Characterization of the Annexin A2 / P11 Complex ............ 50
3.1 Purification of A2t from Pig Small Intestine ...............................................................50
3.1.1 Preparation of Small Intestines .......................................................................................... 50
3.1.2 Sample Preparation for Chromatographic Separation ....................................................... 51
3.1.3 Protein Purification by Fast Protein Liquid Chromatography ............................................. 52
II
3.1.4 Mass Spectrometric Identification of ANXA2, P11, and Co-Purified Proteins.................... 53
3.2 Characterization of A2t...........................................................................