Crystallization and structural characterization of protein complexes involved in the energy metabolism of Yarrowia lipolytica [Elektronische Ressource] / von Blanka Ksymena Wrzesniewska

Blanka Vavakova

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 32
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Crystallization and structural characterization of protein
complexes involved in the energy metabolism of
Yarrowia lipolytica



Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich Biochemie, Chemie und Pharmazie
der Goethe-Universität
in Frankfurt am Main




von
Blanka Ksymena Wrzesniewska
aus Opole (Polen)





Frankfurt am Main 2009
(D30)

























Vom Fachbereich Biochemie, Chemie und Pharmazie der Goethe-Universität als Dissertation
angenommen.


Dekan: Prof. Dr. Dieter Steinhilber

Gutachter: Prof. Dr. Bernd Ludwig
Prof. Dr. Ulrich Brandt




Datum der Disputation: 27.04.2009





Die vorliegende Arbeit wurde in der Zeit von September 2004 bis September 2008 im Gus-
tav-Embden Zentrum der Biologischen Chemie, Molekulare Bioenergetik, des Universitäts-
klinikums der Goethe-Universität in Frankfurt am Main unter Anleitung von Prof. Dr. Ulrich
Brandt durchgeführt.

Acknowledgements
I would like to sincerely thank Prof. Ulrich Brandt who offered me a warm welcome in the
Molecular Bioenergetics group, provided with financial support and necessary research facili-
ties in his lab. For his excellent scientific supervision, advice, continuous support and passion
for science, I am greatly thankful.

I am grateful to Prof. Bernd Ludwig for taking the responsibility of external supervision.

I would like to thank Dr. Volker Zickermann for outstanding scientific guidance, valuable
suggestions and many fruitful discussions, as well as for critical reading of the manuscript.

I thank Prof. Carola Hunte for support in crystallography and fruitful cooperation.

I would also like to thank
Prof. Hermann Schägger for scientific discussions and helpful suggestions.
Dr. Stefan Kerscher for scientific support in the molecular biology field.
Dr. Ilka Wittig for introduction to cell culture techniques as well as mass spectrometry.
Maja Tocilescu for a helpful hand, lots of stimulating discussions and for being a dear friend.
Karin Siegmund and Andrea Duchene for excellent technical assistance, encouragement and
lots of heart.
Dr. Klaus Zwicker for valuable suggestions and kindness.
Esther Nübel for ‘all that jazz…’
Lucie Sokolova for performing the LILBID experiments.
Dipl.-Ing. Gudrun Beyer for valuable hints in the molecular biology field.
Nicola Crosetto for introduction to SPR techniques.
Andrea Böttcher for support in IT-related issues.

I am very thankful to all group members for creating a nice working environment.

Last, but not least, I would like to thank Kris, without whom I wouldn’t have been, where I
am now.
Finally, I am grateful to my family Mom, Dad, Anetka, Mariusz, Michal, Marta, Sandra, Iwo-
na, Zbyszek who always believed in me.















Mojej rodzinie,Krzysiowi




Crystallization and structural characterization of protein
complexes involved in the energy metabolism of
Yarrowia lipolytica






Part I
Fab co-complexes of proton pumping
NADH:ubiquinone oxidoreductase (complex I)



Part II
UDP-glucose pyrophosphorylase

INDEX OF CONTENTS


Part I-General introduction..............................................................................................................1
1 Complex I – mysterious molecular machinery ........................................................................2
1.1 Subunit composition of complex I ......................................................................................3
1.2 Functional modules of complex I ........................................................................................4
1.3 Structure of complex I ........................................................................................................5
1.4 Energy coupling hypothesis ................................................................................................7
2 Crystallization of membrane proteins......................................................................................8
2.1 Selection of a suitable detergent .........................................................................................8
2.2 Types of membrane protein crystals ...................................................................................9
2.3 Antibody fragments for co-crystallization with membrane proteins ................................... 11
2.3.1 Fab and Fv fragments............................................................................................... 13
2.4 Crystallization of membrane proteins with anti-body fragments ........................................ 15
2.5 Enlargement of the polar surface – alternative to the antibody fragments .......................... 17
Part I-Results .................................................................................................................................. 18
3 Generation of antibody Fab fragments .................................................................................. 19
3.1 Production and purification of monoclonal antibodies....................................................... 19
3.2 Generation of proteolytic antibody fragments ................................................................... 22
3.2.1 Crystalline papain .................................................................................................... 23
3.2.2 Immobilized ficin .................................................................................................... 25
3.2.3 Immobilized papain ................................................................................................. 26
3.3 Purification of functional Fab fragments ........................................................................... 27
3.3.1 Cation exchange ...................................................................................................... 27
3.3.2 Anion exchange ....................................................................................................... 28
3.3.3 Immobilized papain digestion mixture as Fab source ................................................ 30
3.3.4 Isolation of pure Fab fragments from the immobilized papain digestion mixture ....... 32
3.3.4.1 Double gel filtration ........................................................................................ 32
3.3.4.2 Protein G spin columns followed by gel filtration ............................................ 33
4 Surface plasmon resonance measurements ............................................................................ 36
4.1 Affinities of the monoclonal antibodies and the Fab fragments to complex I ..................... 36
4.2 Influence of conformation on the binding of the monoclonal antibodies to complex I........ 36
5 Preparation of the CI/Fab co-complexes ............................................................................... 38
5.1 CI/1F5 Fab co-complexes ................................................................................................. 38
I
INDEX OF CONTENTS


5.2 CI/Fab 44G10 co-complexes ............................................................................................ 39
5.3 CI/Fab 31A8 co-complexes .............................................................................................. 40
5.4 Activity assays and phosphate determination OF CI/Fab co-complexes ............................ 42
6 Crystallization of CI/Fab co-complexes ................................................................................ 44
6.1 CI/Fab 31A8 co-complex crystallization ........................................................................... 44
6.2 CI/Fab 44G10 co-complexes crystallization ...................................................................... 47
6.3 CI/Fab 1F5 co-complexes crystallization .......................................................................... 49
6.3.1 Initial crystallization conditions ............................................................................... 49
6.3.2 Optimization of crystallization conditions ................................................................ 51
6.3.2.1 Additive screen................................................................................................ 51
6.3.2.2 Oils in crystallization of CI/Fab co-complexes ................................................. 52
6.3.2.3 Seeding ........................................................................................................... 53
6.3.2.4 Lipids and the crystals quality .......................................................................... 53
6.3.2.5 Temperature .................................................................................................... 54
6.3.2.6 Crystal growth using pH gradient...........................